Human tissue inhibitor of metalloproteinase-4

ABSTRACT

A human tissue inhibitor of metalloproteinases-4 polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques. Also disclosed are methods for utilizing such polypeptide for the treatment of diseases, including arthritis and cancer. Antagonists against such polypeptides and their use as a therapeutic to resorb scar tissue are also disclosed. Diagnostic assays for detecting levels of human TIMP-4 protein and mutations in human TMP-4 nucleic acid sequence are also disclosed.

[0001] This application is a Continuation-in-Part of U.S. applicationSer. No. 09/387,525, filed Sep. 1, 1999, which is a Continuation of U.S.application Ser. No. 08/463,261, filed Jun. 5, 1995, which is acontinuation-in-part of PCT/US94/14498, filed Dec. 13, 1994 (filed inEnglish), each of which are hereby incorporated by reference in theirentireties. This application also claims benefit under 35 U.S.C. §119(e), of U.S. Provisional Application Nos. 60/217,419, filed Jul. 11,2000, and No. 60/220,829, filed Jul. 26, 2000, each of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides, polypeptides, and antibodies, as well as the productionof such polynucleotides and polypeptides. More particularly, thepolypeptides of the present invention are human tissue inhibitor ofmetalloproteinase-4 polypeptides, hereinafter referred to as “humanTIMP-4”. The invention also relates to inhibiting the action of suchpolypeptides.

BACKGROUND OF THE INVENTION

[0003] The extracellular matrix is a complex structure that containscollagen, proteoglycan, glycosaminoglycan, glycoproteins (fibronectin,chondronectin, laminin) and in some tissues, elastin (Hay, E. D., J.Cell Biol., 91:205-223 (1981)).

[0004] Matrix metalloproteinases (MMP's) constitute the major group ofzinc-binding endopeptidases that degrade extracellular matrix proteins,for example connective tissue, collagen and gelatin, during remodelingof connective tissue during normal physiological and some pathologicalprocesses. The unrestrained activity of MMP's may result in extensivetissue damage, and these enzymes have been implicated in a variety ofdisease processes, including tumor cell invasion, tumor angiogenesis andrheumatoid arthritis (Okada, Y., et al., J. Biol. Chem., 261:14245-14255(1986)). The MMP's are secreted from cells as inactive zymogens andtheir activity in the extracellular environment is regulated by variousactivators and inhibitors (Matrisian, L. M., Trends Genet., 6:121-125(1990)).

[0005] Regulation of metalloproteinase-mediated proteolysis may occur bynaturally occurring inhibitor proteins, such as tissue inhibitor ofmetalloproteinase (TIMP). The balance between the production andactivation of the MMP's, and their inhibition by natural inhibitors suchas TIMP, determines, in both physiological and pathological conditions,whether connective tissue is degraded.

[0006] MMP's include a number of proteases, exemplified by interstitial(type I) collagenase itself, the stromelysins (also known asproteoglycanases or transins), fibroblast and polymorphonuclearleukocyte gelatinases (also known as collagen-IV-ases), and pump-1(putative metalloproteases 1, uterine metalloproteases) [Goldberg et al,J. Biol. Chem. 2610:6600 (1986); Whitham et al, Biochem. J. 240:913(1986); Breathnach et al, Nucleic Acids Res., 15:1139 (1987); Muller etal, Biochem. J., 253:187 (1988); Collier et al, J. Biol. Chem., 263:6579(1988); Murphy et al, Biochem. J., 258:463 (1989); Quantin et al,Biochem. (N.Y.), 28:5327 (1989); Birkedal-Hansen, J. Oral Pathol.,17:445 (1988)].

[0007] In general, the mammalian family of proteases has one or more ofthe following properties: (a) optimal proteolytic activity aroundneutral pH; (b) dependence of the enzyme's activity on the presence ofzinc, as evident by the loss of activity on treatment with divalentmetal ion chelators, such as 1.10 phenanthroline (preferential chelationof zinc), or EDTA (less restricted chelating properties; EDTA and EGTAalso contribute to enzyme inactivation via chelation of calcium ionsrequired for enzyme stability); (c) inhibition by TIMPs; (d) absence ofsignificant inhibition by known inhibitors of other families of neutral,zinc-containing metalloproteases, such as thermolysis,angiotensin-converting enzyme and ‘enkephalinases’; and (e) biosynthesisand secretion as latent precursor forms (zymogens), requiringextracellular activation. Activation has been achieved by a number ofendoproteases, organomercurials and chaotropic agents.

[0008] In general, members of the family of neutral metalloproteaseenzymes have distinctive substrate specificities. Thus, collagenase typeI is unique in its ability to cleave a specific peptide bond within thenatural fibrils of the interstitial collagens (e.g. types I, II andIII). The gelatinases are only poorly active on these collagens, but areable to degrade denatured interstitial collagens, as well as thenon-fibrillar collagens, e.g. type IV, such as are found in the basementmembrane. Pump 1 has been reported to act preferentially on denaturedcollagens (gelatins), though its profile differs from that of thestromelysins or the collagenases type IV. Both the stromelysins and thegelatinases are also capable of degrading non-collagenous structuralproteins, such as the core protein of proteoglycan and elastin.Macromolecules involved in cell-to-substratum and cell-to-cellinteractions, such as laminin and fibronectin, are also susceptible todegradation by several of these metalloproteases.

[0009] Enzymes of this family are produced by synovial and skinfibroblasts, chondrocytes, peripheral mononuclear cells, keratinocytesand gingival tissue, as well as existing within granule storage vesiclesin polymorphonuclear leukocytes (PMNLs).

[0010] Current information suggests that there is a family ofmetalloproteinase inhibitors which comprises TIMP-1 (tissue inhibitor ofmetalloproteinases-1); TIMP-2; human TIMP-3 which has been cloned,expressed and mapped to human chromosome 22; and chicken tissueinhibitor of metalloproteinase (ChIMP-5). The polypeptide of the presentinvention has been putatively identified as a novel human TIMPpolypeptide based on amino acid sequence homology.

SUMMARY OF THE INVENTION

[0011] In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide which is human TIMP-4, as well asbiologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof.

[0012] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding human TIMP-4,including mRNA's, DNA's, cDNA's, genomic DNA as well as biologicallyactive and diagnostically or therapeutically useful fragments, analogsand derivatives thereof.

[0013] In accordance with yet a further aspect of the present invention,there is provided a process for producing such polypeptide byrecombinant techniques which comprises culturing recombinant prokaryoticand/or eukaryotic host cells, containing a human TIMP-4 nucleic acidsequence under conditions promoting expression of protein and subsequentrecovery of said protein.

[0014] In accordance with yet a further aspect of the present invention,there is provided a method for treating conditions which are related toinsufficient human TIMP-4 activity which comprises administering to apatient in need thereof a pharmaceutical composition containing thehuman TIMP-4 protein of the invention which is effective to supplement apatient's endogenous human TIMP-4 and thereby alleviate said conditionswhich include, for example, arthritic diseases such as rheumatoid andosteoarthritis, soft tissue rheumatism, polychondritis and tendonitis;bone resorption diseases, such as osteoporosis, Paget's disease,hyperparathyroidism and cholesteatoma; the enhanced collagen destructionthat occurs in association with diabetes; the recessive classes ofdystrophic epidermolysis bullosa; periodontal disease, alveolitis andrelated consequences of gingival production of collagenase; cornealulceration; ulceration of the skin and gastrointestinal tract andabnormal wound healing; post-operative conditions in which collagenaselevels are raised; cancer by blocking the destruction of tissue basementmembranes leading to cancer metastasis; demyelinating diseases of thecentral and peripheral nervous systems; asthma; glomerulosclerosis;septic shock and infection; and psoriasis.

[0015] In accordance with yet a further aspect of the present invention,there is provided a method for treating or preventing restenosis, whichcomprises administering to a patient in need thereof a pharmaceuticalcomposition containing the human TIMP-4 protein of the invention whichis effective to treat or prevent restenosis.

[0016] In accordance with yet a further aspect of the present invention,there is provided an antibody against such polypeptides.

[0017] In accordance with yet another aspect of the present invention,there are provided nucleic acid probes comprising nucleic acid moleculesof sufficient length to specifically hybridize to human TIMP-4sequences.

[0018] In accordance with yet another aspect of the present invention,there are provided antagonists to such polypeptides which may beemployed for therapeutic purposes, for example, for remodeling andrepairing tissue and for destruction of scar tissue.

[0019] In accordance with another aspect of the present invention, thereare provided diagnostic assays for detecting diseases related tomutations in human TIMP-4 sequences and over-expression of thepolypeptide.

[0020] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

[0021] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0022] FIGS. 1A-B shows the cDNA sequence and corresponding deducedamino acid sequence of the full-length human TIMP-4 polypeptide. Thestandard one-letter abbreviations for amino acids are used. Sequencingwas performed using a 373 Automated DNA sequencer (Applied Biosystems,Inc.). Sequencing accuracy is predicted to be greater than 97% accurate.

[0023] FIGS. 2A-B is an amino acid sequence comparison between thepolypeptide of the present invention and other human TIMP polypeptides.

[0024] FIGS. 3A-F shows the adenoviral plasmid maps used in the genetherapy experiments described in Example 7.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] In accordance with an aspect of the present invention, there isprovided an isolated nucleic acid (polynucleotide) which encodes for themature polypeptide having the deduced amino acid sequence of FIGS. 1A-Bor for the mature polypeptide encoded by the cDNA of the clone depositedas ATCC Deposit No. 75946 on Nov. 11, 1994.

[0026] A polynucleotide encoding a polypeptide of the present inventionmay be obtained from an early stage human brain. This contains an openreading frame and coding of protein of 224 amino acid residues of whichapproximately the first 29 residues represent the leader sequence suchthat the mature protein comprises 195 amino acid residues. Thepolynucleotide of this invention was discovered in a cDNA libraryderived from an early stage human brain. The protein exhibits thehighest degree of homology to Human TIMP-2 with 48% identity and 72%similarity over a 136 amino acid stretch. Human TIMP-4 has the signature12 cysteine amino acids, which are conserved in all members of the TIMPfamily. The 12 cysteine residues are all disulfide-linked in TIMP-1 andTIMP-2. This evidence strongly suggests that the polypeptide of thepresent invention is a novel member of the TIMP family.

[0027] The polynucleotide of the present invention may be in the form ofRNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in FIGS. 1A-B or that of thedeposited clone or may be a different coding sequence which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same, mature polypeptide as the DNA of FIGS. 1A-B orthe deposited cDNA.

[0028] The polynucleotide which encodes for the mature polypeptide ofFIGS. 1A-B or for the mature polypeptide encoded by the deposited cDNAmay include: only the coding sequence for the mature polypeptide; thecoding sequence for the mature polypeptide and additional codingsequence such as a leader or secretory sequence or a proproteinsequence; the coding sequence for the mature polypeptide (and optionallyadditional coding sequence) and non-coding sequence, such as introns ornon-coding sequence 5′ and/or 3′ of the coding sequence for the maturepolypeptide.

[0029] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0030] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptide having the deduced amino acidsequence of FIGS. 1A-B or the polypeptide encoded by the cDNA of thedeposited clone. The variant of the polynucleotide may be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

[0031] Thus, the present invention includes polynucleotides encoding thesame mature polypeptide as shown in FIGS. 1A-B or the same maturepolypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIGS. 1A-B or the polypeptideencoded by the cDNA of the deposited clone. Such nucleotide variantsinclude deletion variants, substitution variants and addition orinsertion variants.

[0032] As hereinabove indicated, the polynucleotide may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIGS. 1A-B or of the coding sequence of the depositedclone. As known in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

[0033] The present invention also includes polynucleotides, wherein thecoding sequence for the mature polypeptide may be fused in the samereading frame to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains. Thus,for example, the polynucleotide of the present invention may encode fora mature protein, or for a protein having a prosequence or for a proteinhaving both a prosequence and a presequence (leader sequence).

[0034] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

[0035] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0036] Fragments of the full length gene of the present invention may beused as a hybridization probe for a cDNA library to isolate the fulllength cDNA and to isolate other cDNAs which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype preferably have at least 30 bases and may contain, for example, 50or more bases. The probe may also be used to identify a cDNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete gene including regulatory and promotorregions, exons, and introns. An example of a screen comprises isolatingthe coding region of the gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or mRNA to determinewhich members of the library the probe hybridizes to.

[0037] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least70%, preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, in oneembodiment, the term “stringent conditions” means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences. The polynucleotides which hybridize to thehereinabove described polynucleotides in a preferred embodiment encodepolypeptides which either retain substantially the same biologicalfunction or activity as the mature polypeptide encoded by the cDNAs ofFIGS. 1A-B (SEQ ID NO:1) or the deposited cDNA(s). In an alternativeembodiment, by “stringent hybridization conditions” is intendedovernight incubation at 42° C. in a solution comprising: 50% formamide,5× SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate(pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1× SSC at about 65° C.

[0038] Alternatively, the polynucleotide may have at least 20 bases,preferably 30 bases, and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which has anidentity thereto, as hereinabove described, and which may or may notretain activity. For example, such polynucleotides may be employed asprobes for the polynucleotide of SEQ ID NO:1, for example, for recoveryof the polynucleotide or as a diagnostic probe or as a PCR primer.

[0039] Thus, the present invention is directed to polynucleotides havingat least a 70% identity, preferably at least 80%, at least 85%, at least90%, and more preferably at least a 95% identity, at least a 96%identity, at least a 97% identity, at least a 98% identity, or at leasta 99% identity, to a polynucleotide which encodes the polypeptide of SEQID NO:2 as well as fragments thereof, which fragments have at least 30bases and preferably at least 50 bases and to polypeptides encoded bysuch polynucleotides. The deposit(s) referred to herein will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Micro-organisms for purposes of PatentProcedure. These deposits are provided merely as convenience to those ofskill in the art and are not an admission that a deposit is requiredunder 35 U.S.C. §112. The sequence of the polynucleotides contained inthe deposited materials, as well as the amino acid sequence of thepolypeptides encoded thereby, are incorporated herein by reference andare controlling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0040] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” or “identity” to a reference nucleotidesequence encoding a TIMP-4 polypeptide is intended that the nucleotidesequence of the polynucleotide is identical to the reference sequenceexcept that the polynucleotide sequence may include up to fivemismatches per each 100 nucleotides of the reference nucleotide sequenceencoding the TIMP-4 polypeptide. In other words, to obtain apolynucleotide having a nucleotide sequence at least 95% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. The reference (query) sequence maybe the entire nucleotide sequence encoding TIMP-4, as shown in FIGS. 1Aand 1B (SEQ ID NO:1) or any TIMP-4 polynucleotide sequence describedherein.

[0041] As a practical matter, whether any particular nucleic acidmolecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identicalto, for instance, the nucleotide sequences shown in FIGS. 1A and 1B, orto the cDNA sequence of the deposited cDNA clone, or fragments thereof,can be determined conventionally using known computer programs such asthe Bestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). Bestfit uses the local homology algorithmof Smith and Waterman to find the best segment of homology between twosequences (Advances in Applied Mathematics 2:482-489 (1981)). When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference nucleotide sequence and that gaps in homology ofup to 5% of the total number of nucleotides in the reference sequenceare allowed.

[0042] In a specific embodiment, the identity between a reference(query) sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, is determinedusing the FASTDB computer program based on the algorithm of Brutlag andcolleagues (Comp. App. Biosci. 6:237-245 (1990)). In a sequencealignment the query and subject sequences are both DNA sequences. An RNAsequence can be compared by converting U's to T's. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB alignment of DNA sequences to calculate percentidentity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, JoiningPenalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5,Gap Size Penalty 0.05, Window Size=500 or the length of the subjectnucleotide sequence, whichever is shorter. According to this embodiment,if the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionis made to the results to take into consideration the fact that theFASTDB program does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.A determination of whether a nucleotide is matched/aligned is determinedby results of the FASTDB sequence alignment. This percentage is thensubtracted from the percent identity, calculated by the above FASTDBprogram using the specified parameters, to arrive at a final percentidentity score. This corrected score is what is used for the purposes ofthis embodiment. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score. For example, a 90 basesubject sequence is aligned to a 100 base query sequence to determinepercent identity. The deletions occur at the 5′ end of the subjectsequence and therefore, the FASTDB alignment does not show amatched/alignment of the first 10 bases at 5′ end. The 10 unpaired basesrepresent 10% of the sequence (number of bases at the 5′ and 3′ ends notmatched/total number of bases in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 bases were perfectly matched the finalpercent identity would be 90%. In another example, a 90 base subjectsequence is compared with a 100 base query sequence. This time thedeletions are internal deletions so that there are no bases on the 5′ or3′ of the subject sequence which are not matched/aligned with the query.In this case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only bases 5′ and 3′ of the subject sequencewhich are not matched/aligned with the query sequence are manuallycorrected for. No other manual corrections are made for the purposes ofthis embodiment.

[0043] The present application is directed to nucleic acid molecules atleast 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to thenucleic acid sequences (i.e., polynucleotides) disclosed herein (e.g.,those disclosed in FIGS. 1A and 1B (SEQ ID NO:1) or to the cDNA sequenceof the deposited clone), irrespective of whether they encode apolypeptide having TIMP-4 functional activity (e.g., biologicalactivity). This is because even where a particular nucleic acid moleculedoes not encode a polypeptide having TIMP-4 activity, one of skill inthe art would still know how to use the nucleic acid molecule, forinstance, as a hybridization probe or a polymerase chain reaction (PCR)primer. Uses of the nucleic acid molecules of the present invention thatdo not encode a polypeptide having TIMP-4 activity include, inter alia,(1) isolating the TIMP-4 gene or allelic variants thereof in a cDNAlibrary; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of theTIMP-4 gene, as described in Verma et al., Human Chromosomes: A Manualof Basic Techniques, Pergamon Press, New York (1988); and Northern Blotanalysis for detecting TIMP-4 mRNA expression in specific tissues.

[0044] Preferred, however, are nucleic acid molecules having sequencesat least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to thenucleic acid sequences disclosed herein (e.g., the nucleotide sequenceshown in FIGS. 1A and 1B (SEQ ID NO:1) and the cDNA sequence of thedeposited clone, or fragments thereof), which do, in fact, encode apolypeptide having TIMP-4 polypeptide functional activity (e.g.,biological activity).

[0045] By “a polypeptide having TIMP-4 functional activity” (e.g.,biological activity) is intended polypeptides exhibiting activitysimilar, but not necessarily identical, to an activity of TIMP-4polypeptides of the invention, as measured in a particular functionalassay. TIMP-4 “functional activities include, but are not limited to,biological activity (e.g., ability to inhibit metalloproteinaseactivity, ability to inhibit the proliferation of cardiac smoothmuscles, ability to inhibit the formation of the inner lining (neotima)of the carotid artery following balloon angioplasty injury),antigenicity [ability to bind (or compete with a TIMP-4 polypeptide forbinding) to an anti-TIMP-4 antibody,], immunogenicity (ability togenerate antibody which binds to a TIMP-4 polypeptide), and ability tobind to a TIMP-4 receptor/ligand. Techniques known in the art may beapplied to routinely determine if polypeptides of the invention exhibitTIMP-4 functional activities (e.g., biological activity (e.g., abilityto inhibit metalloproteinases (e.g., metalloproteinase 1, 2, 3, 7, and9))).

[0046] In specific embodiments, the polynucleotides of the inventioncomprise, or alternatively consist of, a polynucleotide sequence that isat least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, or 200, contiguous nucleotides of SEQ ID NO:1.Polypeptides encoded by these polynucleotides are also encompassed bythe invention.

[0047] In specific embodiments, the polynucleotides of the inventioncomprise, or alternatively consist of, a polynucleotides sequenceencoding a polypeptide sequence that is at least 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200,contiguous amino acids of SEQ ID NO:2. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention.

[0048] In specific embodiments, the polynucleotides of the inventioncomprise, or alternatively consist of, a nucleotide sequence encoding apolypeptide sequence selected from the group: (a) a polypeptide havingthe amino acid sequence of amino acids 22 to 28 of SEQ ID NO:2; and (b)a polypeptide having the amino acid sequence of amino acids 34 to 40 ofSEQ ID NO:2. Polypeptides encoded by these polynucleotides are alsoencompassed by the invention.

[0049] In specific embodiments, the polynucleotides of the inventioncomprise, or alternatively consist of, a nucleotide sequence encoding apolypeptide sequence selected from the group: (a) a polypeptide havingthe amino acid sequence of amino acids 1 to 72 of SEQ ID NO:2; (b) apolypeptide having the amino acid sequence of amino acids 73 to 127 ofSEQ ID NO:2; (c) a polypeptide having the amino acid sequence of aminoacids 128 to 176 of SEQ ID NO:2; and (d) a polypeptide having the aminoacid sequence of amino acids 1 to 176 of SEQ ID NO:2. Polypeptidesencoded by these polynucleotides are also encompassed by the invention.

[0050] In specific embodiments, the polynucleotides of the inventioncomprise, or alternatively consist of, a nucleotide sequence encoding apolypeptide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to a polypeptide sequence selected from thegroup: (a) a polypeptide having the amino acid sequence of amino acids 1to 72 of SEQ ID NO:2; (b) a polypeptide having the amino acid sequenceof amino acids 73 to 127 of SEQ ID NO:2; (c) a polypeptide having theamino acid sequence of amino acids 128 to 176 of SEQ ID NO:2; and (d) apolypeptide having the amino acid sequence of amino acids 1 to 176 ofSEQ ID NO:2. Polypeptides encoded by these polynucleotides are alsoencompassed by the invention.

[0051] The present invention further relates to a human TIMP-4polypeptide which has the deduced amino acid sequence of FIGS. 1A-B orwhich has the amino acid sequence encoded by the deposited cDNA, as wellas fragments, analogs and derivatives of such polypeptide.

[0052] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide of FIGS. 1A-B or that encoded by the deposited cDNA,means a polypeptide which retains essentially the same biologicalfunction or activity as such polypeptide. Thus, an analog includes aproprotein which can be activated by cleavage of the proprotein portionto produce an active mature polypeptide.

[0053] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

[0054] The fragment, derivative or analog of the polypeptide of FIGS.1A-B or that encoded by the deposited cDNA may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

[0055] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0056] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0057] In specific embodiments, the polypeptides of the inventioncomprise, or alternatively consist of, an amino acid sequence that is atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, or 200, contiguous amino acids of SEQ ID NO:2.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0058] In specific embodiments, the polypeptides of the inventioncomprise, or alternatively consist of, a polypeptide selected from thegroup: (a) a polypeptide having the amino acid sequence of amino acids 1to 72 of SEQ ID NO:2; (b) a polypeptide having the amino acid sequenceof amino acids 73 to 127 of SEQ ID NO:2; (c) a polypeptide having theamino acid sequence of amino acids 128 to 176 of SEQ ID NO:2; and (d) apolypeptide having the amino acid sequence of amino acids 1 to 176 ofSEQ ID NO:2. Poynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0059] In specific embodiments, the polypeptides of the inventioncomprise, or alternatively consist of, a polypeptide selected from thegroup: (a) a polypeptide having the amino acid sequence of amino acids22 to 28 of SEQ ID NO:2; and (b) a polypeptide having the amino acidsequence of amino acids 34 to 40 of SEQ ID NO:2. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

[0060] Preferred polypeptide fragments of the invention include thesecreted protein as well as the mature form. Further preferredpolypeptide fragments include the secreted protein or the mature formhaving a continuous series of deleted residues from the amino or thecarboxy terminus, or both. Poynucleotides encoding these polypeptidesare also encompassed by the invention.

[0061] Accordingly, polypeptide fragments include the secreted TIMP-4protein as well as the mature form. Further preferred polypeptidefragments include the secreted TIMP-4 protein or the mature form havinga continuous series of deleted residues from the amino or the carboxyterminus, or both. For example, any number of amino acids, ranging from1-29 of the TIMP-4 sequence disclosed in FIGS. 1A-B, can be deleted fromthe amino terminus of either the secreted TIMP-4 polypeptide or themature form. Similarly, any number of amino acids, ranging from 1-30 ofthe TIMP-4 sequence disclosed in FIGS. 1A-B, can be deleted from thecarboxy terminus of the secreted TIMP-4 protein or mature form.Furthermore, any combination of the above amino and carboxy terminusdeletions are preferred. Similarly, polynucleotides encoding thesepolypeptide fragments are also preferred. Particularly, N-terminaldeletions of the TIMP-4 polypeptide can be described by the generalformula m-224, where m is an integer from 2-218, where m corresponds tothe position of the amino acid residue identified in FIGS. 1A-B. More inparticular, the invention provides polypeptides comprising, oralternatively consisting of, an amino acid sequence selected from: P-2to P-224; G-3 to P-224; S-4 to P-224; P-5 to P-224; R-6 to P-224; P-7 toP-224; A-8 to P-224; P-9 to P-224; S-10 to P-224; W-11 to P-224; V-12 toP-224; L-13 to P-224; L-14 to P-224; L-15 to P-224; R-16 to P-224; L-17to P-224; L-18 to P-224; A-19 to P-224; L-20 to P-224; L-21 to P-224;R-22 to P-224; P-23 to P-224; P-24 to P-224; G-25 to P-224; L-26 toP-224; G-27 to P-224; E-28 to P-224; A-29 to P-224; C-30 to P-224; S-31to P-224; C-32 to P-224; A-33 to P-224; P-34 to P-224; A-35 to P-224;H-36 to P-224; P-37 to P-224; Q-38 to P-224; Q-39 to P-224; H-40 toP-224; 1-41 to P-224; C-42 to P-224; H-43 to P-224; S-44 to P-224; A-45to P-224; L-46 to P-224; V-47 to P-224; 1-48 to P-224; R-49 to P-224;A-50 to P-224; K-51 to P-224; I-52 to P-224; S-53 to P-224; S-54 toP-224; E-55 to P-224; K-56 to P-224; V-57 to P-224; V-58 to P-224; P-59to P-224; A-60 to P-224; S-61 to P-224; A-62 to P-224; D-63 to P-224;P-64 to P-224; A-65 to P-224; D-66 to P-224; T-67 to P-224; E-68 toP-224; K-69 to P-224; M-70 to P-224; L-71 to P-224; R-72 to P-224; Y-73to P-224; E-74 to P-224; I-75 to P-224; K-76 to P-224; Q-77 to P-224;1-78 to P-224; K-79 to P-224; M-80 to P-224; F-81 to P-224; K-82 toP-224; G-83 to P-224; F-84 to P-224; E-85 to P-224; K-86 to P-224; V-87to P-224; K-88 to P-224; D-89 to P-224; V-90 to P-224; Q-91 to P-224;Y-92 to P-224; 1-93 to P-224; Y-94 to P-224; T-95 to P-224; P-96 toP-224; F-97 to P-224; D-98 to P-224; S-99 to P-224; S-100 to P-224;L-101 to P-224; C-102 to P-224; G-103 to P-224; V-104 to P-224; K-105 toP-224; L-106 to P-224; E-107 to P-224; A-108 to P-224; N-109 to P-224;S-110 to P-224; Q-111 to P-224; K-112 to P-224; Q-113 to P-224; Y-114 toP-224; L-115 to P-224; L-116 to P-224; T-117 to P-224; G-118 to P-224;Q-119 to P-224; V-120 to P-224; L-121 to P-224; S-122 to P-224; D-123 toP-224; G-124 to P-224; K-125 to P-224; V-126 to P-224; F-127 to P-224;I-128 to P-224; H-129 to P-224; L-130 to P-224; C-131 to P-224; N-132 toP-224; Y-133 to P-224; I-134 to P-224; E-135 to P-224; P-136 to P-224;W-137 to P-224; E-138 to P-224; D-139 to P-224; L-140 to P-224; S-141 toP-224; L-142 to P-224; V-143 to P-224; Q-144 to P-224; R-145 to P-224;E-146 to P-224; S-147 to P-224; L-148 to P-224; N-149 to P-224; H-150 toP-224; H-151 to P-224; Y-152 to P-224; H-153 to P-224; L-154 to P-224;N-155 to P-224; C-156 to P-224; G-157 to P-224; C-158 to P-224; Q-159 toP-224; I-160 to P-224; T-161 to P-224; T-162 to P-224; C-163 to P-224;Y-164 to P-224; T-165 to P-224; V-166 to P-224; P-167 to P-224; C-168 toP-224; T-169 to P-224; I-170 to P-224; S-171 to P-224; A-172 to P-224;P-173 to P-224; N-174 to P-224; E-175 to P-224; C-176 to P-224; L-177 toP-224; W-178 to P-224; T-179 to P-224; D-180 to P-224; W-181 to P-224;L-182 to P-224; L-183 to P-224; E-184 to P-224; R-185 to P-224; K-186 toP-224; L-187 to P-224; Y-188 to P-224; G-189 to P-224; Y-190 to P-224;Q-191 to P-224; A-192 to P-224; Q-193 to P-224; H-194 to P-224; Y-195 toP-224; V-196 to P-224; C-197 to P-224; M-198 to P-224; K-199 to P-224;H-200 to P-224; V-201 to P-224; D-202 to P-224; G-203 to P-224; T-204 toP-224; C-205 to P-224; S-206 to P-224; W-207 to P-224; Y-208 to P-224;R-209 to P-224; G-210 to P-224; H-211 to P-224; L-212 to P-224; P-213 toP-224; L-214 to P-224; R-215 to P-224; K-216 to P-224; E-217 to P-224;F-218 to P-224; and V-219 to P-224; of the amino acid sequence in FIGS.1A-B (the amino acid position in FIGS. 1A-B correspond to that of thesequence in SEQ ID NO:2 plus 29). The present application is alsodirected to polypeptides comprising, or alternatively, consisting of, anamino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%identical to a polypeptide described above. The present invention alsoencompasses the above polypeptide sequences fused to a heterologouspolypeptide sequence. Polynucleotides encoding these polypeptides arealso encompassed by the invention.

[0062] Also as mentioned above, even if deletion of one or more aminoacids from the C-terminus of a protein results in modification of lossof one or more biological functions of the protein, other functionalactivities (e.g., biological activities, ability to bind TIMP-4 ligand)may still be retained. For example, the ability of the shortened TIMP-4mutein to induce and/or bind to antibodies which recognize the completeor mature forms of the polypeptide generally will be retained when lessthan the majority of the residues of the complete or mature polypeptideare removed from the C-terminus. Whether a particular polypeptidelacking C-terminal residues of a complete polypeptide retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thatan TIMP-4 mutein with a large number of deleted C-terminal amino acidresidues may retain some biological or immunogenic activities. In fact,peptides composed of as few as six TIMP-4 amino acid residues may oftenevoke an immune response.

[0063] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the carboxy terminus of theamino acid sequence of the TIMP-4 polypeptide shown in FIG. 1 (SEQ IDNO:2), as described by the general formula 1-n, where n is an integerfrom 6-219, where n corresponds to the position of amino acid residueidentified in FIGS. 1A-B. More in particular, the invention providespolypeptides comprising, or alternatively consisting of, an amino acidsequence selected from: M-1 to Q-223; M-1 to V-222; M-1 to 1-221; M-1 toD-220; M-1 to V-219; M-1 to F-21; M-1 to E-217; M-1 to K-216; M-1 toR-215; M-1 to L-214; M-1 to P-213; M-1 to L-212; M-1 to H-211; M-1 toG-210; M-1 to R-209; M-1 to Y-208; M-1 to W-207; M-1 to S-206; M-1 toC-205; M-1 to T-204; M-1 to G-203; M-1 to D-202; M-1 to V-201; M-1 toH-200; M-1 to K-199; M-1 to M-198; M-1 to C-197; M-1 to V-196; M-1 toY-195; M-1 to H-194; M-1 to Q-193; M-1 to A-192; M-1 to Q-191; M-1 toY-190; M-1 to G-189; M-1 to Y-188; M-1 to L-187; M-1 to K-186; M-1 toR-185; M-1 to E-184; M-1 to L-183; M-1 to L-182; M-1 to W-181; M-1 toD-180; M-1 to T-179; M-1 to W-178; M-1 to L-177; M-1 to C-176; M-1 toE-175; M-1 to N-174; M-1 to P-173; M-1 to A-172; M-1 to S-171; M-1 to1-170; M-1 to T-169; M-1 to C-168; M-1 to P-167; M-1 to V-166; M-1 toT-165; M-1 to Y-164; M-1 to C-163; M-1 to T-162; M-1 to T-161; M-1 toI-160; M-1 to Q-159; M-1 to C-158; M-1 to G-157; M-1 to C-156; M-1 toN-155; M-1 to L-154; M-1 to H-153; M-1 to Y-152; M-1 to H-151; M-1 toH-150; M-1 to N-149; M-1 to L-148; M-1 to S-147; M-1 to E-146; M-1 toR-145; M-1 to Q-144; M-1 to V-143; M-1 to L-142; M-1 to S-141; M-1 toL-140; M-1 to D-139; M-1 to E-138; M-1 to W-137; M-1 to P-136; M-1 toE-135; M-1 to 1-134; M-1 to Y-133; M-1 to N-132; M-1 to C-131; M-1 toL-130; M-1 to H-129; M-1 to 1-128; M-1 to F-127; M-1 to V-126; M-1 toK-125; M-1 to G-124; M-1 to D-123; M-1 to S-122; M-1 to L-121; M-1 toV-120; M-1 to Q-119; M-1 to G-118; M-1 to T-117; M-1 to L-116; M-1 toL-115; M-1 to Y-114; M-1 to Q-113; M-1 to K-112; M-1 to Q-111; M-1 toS-110; M-1 to N-109; M-1 to A-108; M-1 to E-107; M-1 to L-106; M-1 toK-105; M-1 to V-104; M-1 to G-103; M-1 to C-102; M-1 to L-101; M-1 toS-100; M-1 to S-99; M-1 to D-98; M-1 to F-97; M-1 to P-96; M-1 to T-95;M-1 to Y-94; M-1 to 1-93; M-1 to Y-92; M-1 to Q-91; M-1 to V-90; M-1 toD-89; M-1 to K-88; M-1 to V-87; M-1 to K-86; M-1 to E-85; M-1 to F-84;M-1 to G-83; M-1 to K-82; M-1 to F-81; M-1 to M-80; M-1 to K-79; M-1 to1-78; M-1 to Q-77; M-1 to K-76; M-1 to 1-75; M-1 to E-74; M-1 to Y-73;M-1 to R-72; M-1 to L-71; M-1 to M-70; M-1 to K-69; M-1 to E-68; M-1 toT-67; M-1 to D-66; M-1 to A-65; M-1 to P-64; M-1 to D-63; M-1 to A-62;M-1 to S-61; M-1 to A-60; M-1 to P-59; M-1 to V-58; M-1 to V-57; M-1 toK-56; M-1 to E-55; M-1 to S-54; M-1 to S-53; M-1 to 1-52; M-1 to K-51;M-1 to A-50; M-1 to R-49; M-1 to 1-48; M-1 to V-47; M-1 to L-46; M-1 toA-45; M-1 to S-44; M-1 to H-43; M-1 to C-42; M-1 to I-41; M-1 to H-40;M-1 to Q-39; M-1 to Q-38; M-1 to P-37; M-1 to H-36; M-1 to A-35; M-1 toP-34; M-1 to A-33; M-1 to C-32; M-1 to S-31; M-1 to C-30; M-1 to A-29;M-1 to E-28; M-1 to G-27; M-1 to L-26; M-1 to G-25; M-1 to P-24; M-1 toP-23; M-1 to R-22; M-1 to L-21; M-1 to L-20; M-1 to A-19; M-1 to L-18;M-1 to L-17; M-1 to R-16; M-1 to L-15; M-1 to L-14; M-1 to L-13; M-1 toV-12; M-1 to W-11; M-1 to S-10; M-1 to P-9; M-1 to A-8; and M-1 to P-7;of FIGS. 1A-B (the amino acid position in FIGS. 1A-B correspond to thatof the sequence in SEQ ID NO:2 plus 29). The present application is alsodirected to polypeptides comprising, or alternatively, consisting of, anamino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%identical to a polypeptide described above. The present invention alsoencompasses the above polypeptide sequences fused to a heterologouspolypeptide sequence. Polynucleotides encoding these polypeptides arealso encompassed by the invention.

[0064] In further embodiments, the present invention encompassespolypeptides comprising, or alternatively consisting of, an epitope ofthe polypeptide having an amino acid sequence of SEQ ID NO:2, or anepitope of the polypeptide sequence encoded by a polynucleotide sequencecontained in deposited clone 75946 or encoded by a polynucleotide thathybridizes to the complement of the sequence of SEQ ID NO:1 or containedin deposited clone 75946 under stringent hybridization conditions asdefined supra. The present invention further encompasses polynucleotidesequences encoding an epitope of a polypeptide sequence of the invention(such as, for example, the sequence disclosed in SEQ ID NO:1),polynucleotide sequences of the complementary strand of a polynucleotidesequence encoding an epitope of the invention, and polynucleotidesequences which hybridize to the complementary strand under stringenthybridization conditions or lower stringency hybridization conditionsdefined supra. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0065] The term “epitopes,” as used herein, refers to portions of apolypeptide having antigenic or immunogenic activity in an animal,preferably a mammal, and most preferably in a human. In a preferredembodiment, the present invention encompasses a polypeptide comprisingan epitope, as well as the polynucleotide encoding this polypeptide. An“immunogenic epitope,” as used herein, is defined as a portion of aprotein that elicits an antibody response in an animal, as determined byany method known in the art, for example, by the methods for generatingantibodies described infra. (See, for example, Geysen et al., Proc.Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,”as used herein, is defined as a portion of a protein to which anantibody can immunospecifically bind its antigen as determined by anymethod well known in the art, for example, by the immunoassays describedherein. Immunospecific binding excludes non-specific binding but doesnot necessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

[0066] Fragments that function as epitopes may be produced by anyconventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

[0067] In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 15, at least20, at least 25, and, most preferably, between about 15 to about 30amino acids. Preferred polypeptides comprising immunogenic or antigenicepitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Antigenicepitopes are useful, for example, to raise antibodies, includingmonoclonal antibodies, that specifically bind the epitope. Antigenicepitopes can be used as the target molecules in immunoassays. (See, forinstance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al.,Science 219:660-666 (1983)).

[0068] Similarly, immunogenic epitopes can be used, for example, toinduce antibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). A preferred immunogenic epitope includes thesecreted TIMP-4 protein. The polypeptides comprising one or moreimmunogenic epitopes may be presented for eliciting an antibody responsetogether with a carrier protein, such as an albumin, to an animal system(such as, for example, rabbit or mouse), or, if the polypeptide is ofsufficient length (at least about 25 amino acids), the polypeptide maybe presented without a carrier. However, immunogenic epitopes comprisingas few as 8 to 10 amino acids have been shown to be sufficient to raiseantibodies capable of binding to, at the very least, linear epitopes ina denatured polypeptide (e.g., in Western blotting).

[0069] Epitope-bearing polypeptides of the present invention may be usedto induce antibodies according to methods well known in the artincluding, but not limited to, in vivo immunization, in vitroimmunization, and phage display methods. See, e.g., Sutcliffe et al.,supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol.,66:2347-2354 (1985). If in vivo immunization is used, animals may beimmunized with free peptide; however, anti-peptide antibody titer may beboosted by coupling the peptide to a macromolecular carrier, such askeyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance,peptides containing cysteine residues may be coupled to a carrier usinga linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),while other peptides may be coupled to carriers using a more generallinking agent such as glutaraldehyde. Animals such as, for example,rabbits, rats, and mice are immunized with either free orcarrier-coupled peptides, for instance, by intraperitoneal and/orintradermal injection of emulsions containing about 100 micrograms ofpeptide or carrier protein and Freund's adjuvant or any other adjuvantknown for stimulating an immune response. Several booster injections maybe needed, for instance, at intervals of about two weeks, to provide auseful titer of anti-peptide antibody that can be detected, for example,by ELISA assay using free peptide adsorbed to a solid surface. The titerof anti-peptide antibodies in serum from an immunized animal may beincreased by selection of anti-peptide antibodies, for instance, byadsorption to the peptide on a solid support and elution of the selectedantibodies according to methods well known in the art.

[0070] As one of skill in the art will appreciate, and as discussedabove, the polypeptides of the present invention comprising animmunogenic or antigenic epitope can be fused to other polypeptidesequences. For example, the polypeptides of the present invention may befused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM),or portions thereof (CH1, CH2, CH3, or any combination thereof andportions thereof) resulting in chimeric polypeptides. Such fusionproteins may facilitate purification and may increase half-life in vivo.This has been shown for chimeric proteins consisting of the first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immunoglobulins. See,e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). IgGFusion proteins that have a disulfide-linked dimeric structure due tothe IgG portion desulfide bonds have also been found to be moreefficient in binding and neutralizing other molecules than monomericpolypeptides or fragments thereof alone. See, e.g., Fountoulakis et al.,J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the aboveepitopes can also be recombined with a gene of interest as an epitopetag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detectionand purification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix-binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

[0071] Additional fusion proteins of the invention may be generatedthrough the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling may be employed to modulate the activities ofpolypeptides of the invention, such methods can be used to generatepolypeptides with altered activity, as well as agonists and antagonistsof the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793;5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr.Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol.16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999);and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of thesepatents and publications are hereby incorporated by reference in itsentirety). In one embodiment, alteration of polynucleotidescorresponding to SEQ ID NO:1 and the polypeptides encoded by thesepolynucleotides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments by homologous or site-specificrecombination to generate variation in the polynucleotide sequence. Inanother embodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide coding apolypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

[0072] The polypeptides of the present invention include the polypeptideof SEQ ID NO:2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptide of SEQ ID NO:2 and more preferably at least90% similarity (more preferably at least 80% identity, or at least 85%identity) to the polypeptide of SEQ ID NO:2 and still more preferably atleast 95% similarity (still more preferably at least 90% identity, atleast 95% identity, at least 96% identity, at least 97% identity, atleast 98% identity, or at least 99% identity ) to the polypeptide of SEQID NO:2 and also include portions of such polypeptides with such portionof the polypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

[0073] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” or “identity” to a reference amino acidsequence of a TIMP-4 polypeptide is intended that the amino acidsequence of the polypeptide is identical to the reference sequenceexcept that the polypeptide sequence may include up to five amino acidalterations per each 100 amino acids of the reference amino acid of theTIMP-4 polypeptide. In other words, to obtain a polypeptide having anamino acid sequence at least 95% identical to a reference amino acidsequence, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

[0074] As a practical matter, whether any particular polypeptide is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the amino acid sequence shown in FIGS. 1A and 1B (SEQ IDNO:2), the amino acid sequence encoded by the deposited cDNA clone, orfragments thereof, can be determined conventionally using known computerprograms such the Bestfit program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,575 Science Drive, Madison, Wis. 53711). When using Bestfit or any othersequence alignment program to determine whether a particular sequenceis, for instance, 95% identical to a reference sequence according to thepresent invention, the parameters are set, of course, such that thepercentage of identity is calculated over the full length of thereference amino acid sequence and that gaps in homology of up to 5% ofthe total number of amino acid residues in the reference sequence areallowed.

[0075] In a specific embodiment, the identity between a reference(query) sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, is determinedusing the FASTDB computer program based on the algorithm of Brutlag etal. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection is made to the results to take into consideration the factthat the FASTDB program does not account for N- and C-terminaltruncations of the subject sequence when calculating global percentidentity. For subject sequences truncated at the N- and C-termini,relative to the query sequence, the percent identity is corrected bycalculating the number of residues of the query sequence that are N- andC-terminal of the subject sequence, which are not matched/aligned with acorresponding subject residue, as a percent of the total bases of thequery sequence. A determination of whether a residue is matched/alignedis determined by results of the FASTDB sequence alignment. Thispercentage is then subtracted from the percent identity, calculated bythe above FASTDB program using the specified parameters, to arrive at afinal percent identity score. This final percent identity score is whatis used for the purposes of this embodiment. Only residues to the N- andC-termini of the subject sequence, which are not matched/aligned withthe query sequence, are considered for the purposes of manuallyadjusting the percent identity score. That is, only query residuepositions outside the farthest N- and C-terminal residues of the subjectsequence. For example, a 90 amino acid residue subject sequence isaligned with a 100 residue query sequence to determine percent identity.The deletion occurs at the N-terminus of the subject sequence andtherefore, the FASTDB alignment does not show a matching/alignment ofthe first 10 residues at the N-terminus. The 10 unpaired residuesrepresent 10% of the sequence (number of residues at the N- andC-termini not matched/total number of residues in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 residues were perfectly matched thefinal percent identity would be 90%. In another example, a 90 residuesubject sequence is compared with a 100 residue query sequence. Thistime the deletions are internal deletions so there are no residues atthe N- or C-termini of the subject sequence which are notmatched/aligned with the query. In this case the percent identitycalculated by FASTDB is not manually corrected. Once again, only residuepositions outside the N- and C-terminal ends of the subject sequence, asdisplayed in the FASTDB alignment, which are not matched/aligned withthe query sequence are manually corrected for. No other manualcorrections are made for the purposes of this embodiment.

[0076] In specific embodiments, the polypeptides of the inventioncomprise, or alternatively consist of, a polypeptitde that is at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to apolypeptide selected from the group: (a) a polypeptide having the aminoacid sequence of amino acids 1 to 72 of SEQ ID NO:2; (b) a polypeptidehaving the amino acid sequence of amino acids 73 to 127 of SEQ ID NO:2;(c) a polypeptide having the amino acid sequence of amino acids 128 to176 of SEQ ID NO:2; and (d) a polypeptide having the amino acid sequenceof amino acids 1 to 176 of SEQ ID NO:2. Poynucleotides encoding thesepolypeptides are also encompassed by the invention.

[0077] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0078] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention. The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0079] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the human TIMP-4 genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

[0080] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0081] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0082] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda PL promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0083] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0084] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0085] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

[0086] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0087] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lac, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0088] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation. (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

[0089] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0090] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0091] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0092] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), alpha-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0093] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0094] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0095] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0096] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0097] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0098] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0099] The human TIMP-4 polypeptides can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (BPLC) can be employed for finalpurification steps.

[0100] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

[0101] Antibodies

[0102] The present invention further relates to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,preferably an epitope, of the present invention (as determined byimmunoassays well known in the art for assaying specificantibody-antigen binding). Antibodies of the invention include, but arenot limited to, polyclonal, monoclonal, multispecific, human, humanizedor chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies of the invention), and epitope-binding fragments of any ofthe above. The term “antibody,” as used herein, refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site thatimmunospecifically binds an antigen. The immunoglobulin molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. In specific embodiments, the immunoglobulinmolecule is IgG1. In other specific embodiments, the immunoglobulinmolecule is IgG4.

[0103] Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine, donkey, shiprabbit, goat, guinea pig, camel, horse, or chicken. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulin and that do not express endogenous immunoglobulins,as described infra and, for example in, U.S. Pat. No. 5,939,598 byKucherlapati et al.

[0104] The antibodies of the present invention may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTpublications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt,et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

[0105] Antibodies of the present invention may be described or specifiedin terms of the epitope(s) or portion(s) of a polypeptide of the presentinvention that they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies thatspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

[0106] Antibodies of the present invention may also be described orspecified in terms of their cross-reactivity. Antibodies that do notbind any other analog, ortholog, or homolog of a polypeptide of thepresent invention are included. Antibodies that bind polypeptides withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. Antibodies that do not bind polypeptides withless than 95%, less than 90%, less than 85%, less than 80%, less than75%, less than 70%, less than 65%, less than 60%, less than 55%, andless than 50% identity (as calculated using methods known in the art anddescribed herein) to a polypeptide of the present invention are alsoincluded in the present invention. Further included in the presentinvention are antibodies that bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity to a polypeptide of theinvention. Preferred binding affinities include those with adissociation constant or Kd less than 5×10⁻²M, 10⁻²M, 5×10⁻³M, 10⁻³M,5×10⁻⁴M, 10⁻⁴M, 5×10⁻⁵M, 10⁻⁵M, 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M,10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M,10⁻¹²M, 5×10⁻³M, 10⁻¹³M, 5×10^(—14)M, 10⁻¹⁴M 5×10⁻¹⁵M, and 10⁻¹⁵M.

[0107] The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%

[0108] Antibodies of the present invention may act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. In specific embodiments, the antagonisticantibodies of the invention increase or enhance metalloproteinaseactivity.). In specific embodiments, antibodies are provided thatincrease or enhance metalloproteinase activity by at least 90%, at least80%, at least 70%, at least 60%, or at least 50% of the activity inabsence of the antibody. In an alternative example, the presentinvention includes antibodies which enhance receptor/ligand interactionswith the polypeptides of the invention either partially or fully. Inspecific embodiments, the agonsitic antibodies of the invention decreaseor reduce metalloproteinase activity. In specific embodiments,antibodies are provided that decrease or reduce metalloproteinaseactivity by at least 90%, at least 80%, at least 70%, at least 60%, orat least 50% of the activity in absence of the antibody. The inventionfeatures both receptor-specific antibodies and ligand-specificantibodies. The invention also features receptor-specific antibodieswhich do not prevent ligand binding but prevent receptor activation.Receptor activation (i.e., signaling) may be determined by techniquesdescribed herein or otherwise known in the art. For example, receptoractivation can be determined by detecting the phosphorylation (e.g.,tyrosine or serine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra). In specific embodiments, antibodies are provided thatenhance or increase ligand or receptor activity by at least 90%, atleast 80%, at least 70%, at least 60%, or at least 50% of the activityin absence of the antibody. In specific embodiments, antibodies areprovided that inhibit ligand or receptor activity by at least 90%, atleast 80%, at least 70%, at least 60%, or at least 50% of the activityin absence of the antibody.

[0109] The invention also features receptor-specific antibodies whichboth prevent ligand binding and receptor activation as well asantibodies that recognize the receptor-ligand complex, and, preferably,do not specifically recognize the unbound receptor or the unboundligand. Likewise, included in the invention are neutralizing antibodieswhich bind the ligand and prevent binding of the ligand to the receptor,as well as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation. The antibodiesmay be specified as agonists, antagonists or inverse agonists forbiological activities comprising the specific biological activities ofthe peptides of the invention disclosed herein. The above antibodyagonists can be made using methods known in the art. See, e.g., PCTpublication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood92(6):1981-1988 (1998); Chen, et al., Cancer Res. 58(16):3668-3678(1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al.,Cancer Res. 58(15):3209-3214 (1998); Yoon, et al., J. Immunol.160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 11(Pt2):237-247(1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997);Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol.Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762(1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al.,Cytokine 8(1):14-20 (1996) (which are all incorporated by referenceherein in their entireties).

[0110] Antibodies of the present invention may be used, for example, butnot limited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

[0111] As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, or toxins. See, e.g., PCT publicationsWO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP396,387.

[0112] The antibodies of the invention include derivatives that aremodified, i.e, by the covalent attachment of any type of molecule to theantibody such that covalent attachment does not prevent the antibodyfrom generating an anti-idiotypic response. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

[0113] The antibodies of the present invention may be generated by anysuitable method known in the art. Polyclonal antibodies to anantigen-of-interest can be produced by various procedures well known inthe art. For example, a polypeptide of the invention can be administeredto various host animals including, but not limited to, rabbits, mice,rats, etc. to induce the production of sera containing polyclonalantibodies specific for the antigen. Various adjuvants may be used toincrease the immunological response, depending on the host species, andinclude but are not limited to, Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

[0114] Monoclonal antibodies can be prepared using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

[0115] Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well-known in the art and arediscussed in detail in Example 6. Briefly, mice can be immunized with apolypeptide of the invention or a cell expressing such peptide. Once animmune response is detected, e.g., antibodies specific for the antigenare detected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by well-knowntechniques to any suitable myeloma cells, for example cells from cellline SP20 available from the ATCC. Hybridomas are selected and cloned bylimited dilution. The hybridoma clones are then assayed by methods knownin the art for cells that secrete antibodies capable of binding apolypeptide of the invention. Ascites fluid, which generally containshigh levels of antibodies, can be generated by immunizing mice withpositive hybridoma clones.

[0116] Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

[0117] Antibody fragments that recognize specific epitopes may begenerated by known techniques. For example, Fab and F(ab′)2 fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

[0118] For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

[0119] As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

[0120] Examples of techniques which can be used to produce single-chainFvs and antibodies include those described in U.S. Pat. Nos. 4,946,778and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991);Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science240:1038-1040 (1988). For some uses, including in vivo use of antibodiesin humans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. A chimeric antibody is amolecule in which different portions of the antibody are derived fromdifferent animal species, such as antibodies having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region. Methods for producing chimeric antibodies are known inthe art. See e.g., Morrison, Science 229:1202 (1985); Oi et al.,BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, whichare incorporated herein by reference in their entireties. Humanizedantibodies are antibody molecules from non-human species antibody thatbinds the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and framework regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

[0121] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which isincorporated herein by reference in its entirety.

[0122] Human antibodies can also be produced using transgenic mice whichare incapable of expressing functional endogenous immunoglobulins, butwhich can express human immunoglobulin genes. For example, the humanheavy and light chain immunoglobulin gene complexes may be introducedrandomly or by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat.Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; and 5,939,598, which are incorporated by reference herein intheir entirety. In addition, companies such as Abgenix, Inc. (Freemont,Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

[0123] Completely human antibodies which recognize a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

[0124] Further, antibodies to the polypeptides of the invention can, inturn, be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

[0125] Polynucleotides Encoding Antibodies

[0126] The invention further provides polynucleotides comprising anucleotide sequence encoding an antibody of the invention and fragmentsthereof. The invention also encompasses polynucleotides that hybridizeunder stringent or lower stringency hybridization conditions, e.g., asdefined supra, to polynucleotides that encode an antibody, preferably,that specifically binds to a polypeptide of the invention, preferably,an antibody that binds to a polypeptide having the amino acid sequenceof SEQ ID NO:2.

[0127] The polynucleotides may be obtained, and the nucleotide sequenceof the polynucleotides determined, by any method known in the art. Forexample, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,BioTechniques 17:242 (1994)), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligation of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

[0128] Alternatively, a polynucleotide encoding an antibody may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the immunoglobulin may be obtained from a suitable source(e.g., an antibody cDNA library, or a cDNA library generated from, ornucleic acid, preferably poly A+ RNA, isolated from, any tissue or cellsexpressing the antibody, such as hybridoma cells selected to express anantibody of the invention) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

[0129] Once the nucleotide sequence and corresponding amino acidsequence of the antibody is determined, the nucleotide sequence of theantibody may be manipulated using methods well known in the art for themanipulation of nucleotide sequences, e.g., recombinant DNA techniques,site directed mutagenesis, PCR, etc. (see, for example, the techniquesdescribed in Sambrook et al., 1990, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, NY, which are both incorporated by reference hereinin their entireties ), to generate antibodies having a different aminoacid sequence, for example to create amino acid substitutions,deletions, and/or insertions.

[0130] In a specific embodiment, the amino acid sequence of the heavyand/or light chain variable domains may be inspected to identify thesequences of the complementarity determining regions (CDRs) by methodsthat are well know in the art, e.g., by comparison to known amino acidsequences of other heavy and light chain variable regions to determinethe regions of sequence hypervariability. Using routine recombinant DNAtechniques, one or more of the CDRs may be inserted within frameworkregions, e.g., into human framework regions to humanize a non-humanantibody, as described supra. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998)for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds a polypeptide of theinvention. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

[0131] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

[0132] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be adaptedto produce single chain antibodies. Single chain antibodies are formedby linking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide. Techniquesfor the assembly of functional Fv fragments in E. coli may also be used(Skerra et al., 1988, Science 242:1038-1041).

[0133] Methods of Producing Antibodies

[0134] The antibodies of the invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or preferably, by recombinant expression techniques.

[0135] Recombinant expression of an antibody of the invention, orfragment, derivative or analog thereof, e.g., a heavy or light chain ofan antibody of the invention, requires construction of an expressionvector containing a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule or a heavy or light chainof an antibody, or portion thereof (preferably containing the heavy orlight chain variable domain), of the invention has been obtained, thevector for the production of the antibody molecule may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Theinvention, thus, provides replicable vectors comprising a nucleotidesequence encoding an antibody molecule of the invention, or a heavy orlight chain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

[0136] The expression vector is transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce an antibody of the invention. Thus,the invention includes host cells containing a polynucleotide encodingan antibody of the invention, or a heavy or light chain thereof,operably linked to a heterologous promoter. In preferred embodiments forthe expression of double-chained antibodies, vectors encoding both theheavy and light chains may be co-expressed in the host cell forexpression of the entire immunoglobulin molecule, as detailed below.

[0137] A variety of host-expression vector systems may be utilized toexpress the antibody molecules of the invention. Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells whichmay, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody molecule of the invention in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing antibodycoding sequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, NSO, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene 45:101; Cockett et al., 1990,Bio/Technology 8:2).

[0138] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

[0139] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The antibody coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

[0140] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the antibody coding sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing the antibody molecule in infectedhosts. (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA81:355-359). Specific initiation signals may also be required forefficient translation of inserted antibody coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBittner et al., 1987, Methods in Enzymol. 153:51-544).

[0141] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

[0142] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the antibody molecule may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

[0143] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler et al.,1977, Cell 11:223), hypoxanthineguanine phosphoribosyltransferase(Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), andadenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genescan be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215); and hygro,which confers resistance to hygromycin (Santerre et al., 1984, Gene30:147). Methods commonly known in the art of recombinant DNA technologywhich can be used are described in Ausubel et al. (eds.), 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CurrentProtocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin etal., 1981, J. Mol. Biol. 150:1, which are incorporated by referenceherein in their entireties.

[0144] The expression levels of an antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol.3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

[0145] The host cell may be co-transfected with two expression vectorsof the invention, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors may contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector may be used which encodes both heavy andlight chain polypeptides. In such situations, the light chain should beplaced before the heavy chain to avoid an excess of toxic free heavychain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad.Sci. USA 77:2197). The coding sequences for the heavy and light chainsmay comprise cDNA or genomic DNA.

[0146] Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

[0147] Antibody Conjugates

[0148] The present invention encompasses antibodies recombinantly fusedor chemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20 or 50 amino acids of the polypeptide) of the present invention togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. The antibodies may bespecific for antigens other than polypeptides (or portion thereof,preferably at least 10, 20 or 50 amino acids of the polypeptide) of thepresent invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etat., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

[0149] The present invention further includes compositions comprisingthe polypeptides of the present invention fused or conjugated toantibody domains other than the variable regions. For example, thepolypeptides of the present invention may be fused or conjugated to anantibody Fc region, or portion thereof. The antibody portion fused to apolypeptide of the present invention may comprise the constant region,hinge region, CH1 domain, CH2 domain, and CH3 domain or any combinationof whole domains or portions thereof. The polypeptides may also be fusedor conjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

[0150] As discussed, supra, the polypeptides of the present inventionmay be fused or conjugated to the above antibody portions to increasethe in vivo half life of the polypeptides or for use in immunoassaysusing methods known in the art. Further, the polypeptides of the presentinvention may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394,827; Traunecker etal., Nature 331:84-86 (1988). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995)0.

[0151] Moreover, the antibodies or fragments thereof of the presentinvention can be fused to marker sequences, such as a peptide tofacilitates their purification. In preferred embodiments, the markeramino acid sequence is a hexa-histidine peptide, such as the tagprovided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

[0152] The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. See,for example, U.S. Pat. No. 4,741,900 for metal ions which can beconjugated to antibodies for use as diagnostics according to the presentinvention. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

[0153] Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion. A cytotoxin orcytotoxic agent includes any agent that is detrimental to cells.Examples include paclitaxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0154] The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

[0155] Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

[0156] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

[0157] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980, which is incorporated herein by reference in itsentirety.

[0158] An antibody, with or without a therapeutic moiety conjugated toit, administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

[0159] Assays For Antibody Binding

[0160] The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

[0161] Immunoprecipitation protocols generally comprise lysing apopulation of cells in a lysis buffer such as RIPA buffer (1% NP-40 orTriton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 Msodium phosphate at pH 7.2, 1% Trasylol) supplemented with proteinphosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin,sodium vanadate), adding the antibody of interest to the cell lysate,incubating for a period of time (e.g., 1-4 hours) at 4° C., addingprotein A and/or protein G sepharose beads to the cell lysate,incubating for about an hour or more at 4° C., washing the beads inlysis buffer and resuspending the beads in SDS/sample buffer. Theability of the antibody of interest to immunoprecipitate a particularantigen can be assessed by, e.g., western blot analysis. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0162] Western blot analysis generally comprises preparing proteinsamples, electrophoresis of the protein samples in a polyacrylamide gel(e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), transferring the protein sample from the polyacrylamide gel toa membrane such as nitrocellulose, PVDF or nylon, blocking the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), blocking themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., 32P or 125I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0163] ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

[0164] The binding affinity of an antibody to an antigen and theoff-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., 3H or 125I) with the antibody of interest in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody ofinterest for a particular antigen and the binding off-rates can bedetermined from the data by scatchard plot analysis. Competition with asecond antibody can also be determined using radioimmunoassays. In thiscase, the antigen is incubated with antibody of interest is conjugatedto a labeled compound (e.g., 3H or 125I) in the presence of increasingamounts of an unlabeled second antibody.

[0165] Therapeutic Uses

[0166] The present invention is further directed to antibody-basedtherapies which involve administering antibodies of the invention to ananimal, preferably a mammal, and most preferably a human, patient fortreating one or more of the described disorders. Therapeutic compoundsof the invention include, but are not limited to, antibodies of theinvention (including fragments, analogs and derivatives thereof asdescribed herein) and nucleic acids encoding antibodies of the invention(including fragments, analogs and derivatives thereof as describedherein). The antibodies of the invention can be used to treat, inhibitor prevent diseases and disorders associated with aberrant expressionand/or activity of a polypeptide of the invention. The treatment and/orprevention of diseases and disorders associated with aberrant expressionand/or activity of a polypeptide of the invention includes, but is notlimited to, alleviating symptoms associated with those diseases anddisorders. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

[0167] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[0168] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

[0169] The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

[0170] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides,including fragments thereof. Preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M,10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M,5×10⁻¹⁵ M, and 10⁻¹⁵ M.

[0171] Gene Therapy

[0172] In a specific embodiment, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit or prevent a disease or disorder associated withaberrant expression and/or activity of a polypeptide of the invention,by way of gene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

[0173] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0174] For general reviews of the methods of gene therapy, see Goldspielet al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

[0175] In a preferred aspect, the compound comprises nucleic acidsequences encoding an antibody, said nucleic acid sequences being partof expression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody nucleic acids(Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935;Zijlstra et al., 1989, Nature 342:435-438). In specific embodiments, theexpressed antibody molecule is a single chain antibody; alternatively,the nucleic acid sequences include sequences encoding both the heavy andlight chains, or fragments thereof, of the antibody.

[0176] Delivery of the nucleic acids into a patient may be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid-carrying vectors, or indirect, in which case, cellsare first transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

[0177] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992(Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/20316dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993(Clarke et al.), WO 93/20221 dated Oct. 14, 1993 (Young)).Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

[0178] In a specific embodiment, viral vectors that contains nucleicacid sequences encoding an antibody of the invention are used. Forexample, a retroviral vector can be used (see Miller et al., 1993, Meth.Enzymol. 217:581-599). These retroviral vectors have been to deleteretroviral sequences that are not necessary for packaging of the viralgenome and integration into host cell DNA. The nucleic acid sequencesencoding the antibody to be used in gene therapy are cloned into one ormore vectors, which facilitates delivery of the gene into a patient.More detail about retroviral vectors can be found in Boesen et al.,1994, Biotherapy 6:291-302, which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141;and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel.3:110-114.

[0179] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5:3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT PublicationWO94/12649; and Wang, et al., 1995, Gene Therapy 2:775-783.

[0180] In cases where an adenovirus is used as an expression vector, theantibody coding sequence of interest may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingthe TIMP-4 molecule in infected hosts. (e.g., see Logan & Shenk, 1984,Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals mayalso be required for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see Bittner et al., 1987, Methods in Enzymol.153:51-544).

[0181] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.204:289-300; U.S. Pat. No. 5,436,146).

[0182] Another approach to gene therapy involves transferring a gene tocells in tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

[0183] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

[0184] The resulting recombinant cells can be delivered to a patient byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

[0185] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

[0186] In a preferred embodiment, the cell used for gene therapy isautologous to the patient.

[0187] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding an antibody are introduced intothe cells such that they are expressible by the cells or their progeny,and the recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598, dated Apr. 28,1994; Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980,Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo ClinicProc. 61:771).

[0188] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription.

[0189] Demonstration of Therapeutic or Prophylactic Activity

[0190] The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

[0191] Therapeutic/Prophylactic Administration and Composition

[0192] The invention provides methods of treatment, inhibition andprophylaxis by administration to a subject of an effective amount of acompound or pharmaceutical composition of the invention, preferably anantibody of the invention. In a preferred aspect, the compound issubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side-effects). The subject ispreferably an animal, including but not limited to animals such as cows,pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal,and most preferably human.

[0193] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid or an immunoglobulin aredescribed above; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

[0194] Various delivery systems are known and can be used to administera compound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of introduction include butare not limited to intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, and oral routes. Thecompounds or compositions may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceutical compoundsor compositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0195] In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

[0196] In another embodiment, the compound or composition can bedelivered in a vesicle, in particular a liposome (see Langer, 1990,Science 249:1527-1533; Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

[0197] In yet another embodiment, the compound or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol.Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989,J.Neurosurg. 71:105). In yet another embodiment, a controlled releasesystem can be placed in proximity of the therapeutic target, i.e., thebrain, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, vol. 2,pp. 115-138 (1984)).

[0198] Other controlled release systems are discussed in the review byLanger (1990, Science 249:1527-1533).

[0199] In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox—like peptide which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

[0200] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of acompound, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0201] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0202] The compounds of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

[0203] The amount of the compound of the invention which will beeffective in the treatment, inhibition and prevention of a disease ordisorder associated with aberrant expression and/or activity of apolypeptide of the invention can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

[0204] For antibodies, the dosage administered to a patient is typically0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, thedosage administered to a patient is between 0.1 mg/kg and 20 mg/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

[0205] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

[0206] Diagnosis and Imaging

[0207] Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity of apolypeptide of the invention. The invention provides for the detectionof aberrant expression of a polypeptide of interest, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared to the standardexpression level is indicative of aberrant expression.

[0208] The invention provides a diagnostic assay for diagnosising adisorder, comprising (a) assaying the expression of the polypeptide ofinterest in cells or body fluid of an individual using one or moreantibodies specific to the polypeptide interest and (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a particulardisorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0209] Antibodies of the invention can be used to assay protein levelsin a biological sample using classical immunohistological methods knownto those of skill in the art (e.g., see Jalkanen, M., et al., J. Cell.Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol.105:3087-3096 (1987)). Other antibody-based methods useful for detectingprotein gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitableantibody assay labels are known in the art and include enzyme labels,such as, glucose oxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I),carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium(⁹⁹Tc); luminescent labels, such as luminol; and fluorescent labels,such as fluorescein and rhodamine, and biotin.

[0210] One aspect of the invention is the detection and diagnosis of adisease or disorder associated with aberrant expression of a polypeptideof the interest in an animal, preferably a mammal and most preferably ahuman. In one embodiment, diagnosis comprises: a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled molecule which specificallybinds to the polypeptide of interest; b) waiting for a time intervalfollowing the administering for permitting the labeled molecule topreferentially concentrate at sites in the subject where the polypeptideis expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled molecule in the subject, such that detection of labeled moleculeabove the background level indicates that the subject has a particulardisease or disorder associated with aberrant expression of thepolypeptide of interest. Background level can be determined by variousmethods including, comparing the amount of labeled molecule detected toa standard value previously determined for a particular system.

[0211] It will be understood in the art that the size of the subject andthe imaging system used will determine the quantity of imaging moietyneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of 99mTc. The labeledantibody or antibody fragment will then preferentially accumulate at thelocation of cells which contain the specific protein. In vivo tumorimaging is described in S. W. Burchiel et al., “Immunopharmacokineticsof Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

[0212] Depending on several variables, including the type of label usedand the mode of administration, the time interval following theadministration for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject and for unbound labeled molecule tobe cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to12 hours. In another embodiment the time interval followingadministration is 5 to 20 days or 5 to 10 days.

[0213] In an embodiment, monitoring of the disease or disorder iscarried out by repeating the method for diagnosing the disease ordisease, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

[0214] Presence of the labeled molecule can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography.

[0215] In a specific embodiment, the molecule is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the molecule is labeled with a fluorescent compoundand is detected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

[0216] Kits

[0217] The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

[0218] In another specific embodiment of the present invention, the kitis a diagnostic kit for use in screening serum containing antibodiesspecific against proliferative and/or cancerous polynucleotides andpolypeptides. Such a kit may include a control antibody that does notreact with the polypeptide of interest. Such a kit may include asubstantially isolated polypeptide antigen comprising an epitope whichis specifically immunoreactive with at least one anti-polypeptideantigen antibody. Further, such a kit includes means for detecting thebinding of said antibody to the antigen (e.g., the antibody may beconjugated to a fluorescent compound such as fluorescein or rhodaminewhich can be detected by flow cytometry). In specific embodiments, thekit may include a recombinantly produced or chemically synthesizedpolypeptide antigen. The polypeptide antigen of the kit may also beattached to a solid support.

[0219] In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which said polypeptideantigen is attached. Such a kit may also include a non-attachedreporter-labeled anti-human antibody. In this embodiment, binding of theantibody to the polypeptide antigen can be detected by binding of thesaid reporter-labeled antibody.

[0220] In an additional embodiment, the invention includes a diagnostickit for use in screening serum containing antigens of the polypeptide ofthe invention. The diagnostic kit includes a substantially isolatedantibody specifically immunoreactive with polypeptide or polynucleotideantigens, and means for detecting the binding of the polynucleotide orpolypeptide antigen to the antibody. In one embodiment, the antibody isattached to a solid support. In a specific embodiment, the antibody maybe a monoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

[0221] In one diagnostic configuration, test serum is reacted with asolid phase reagent having a surface-bound antigen obtained by themethods of the present invention. After binding with specific antigenantibody to the reagent and removing unbound serum components bywashing, the reagent is reacted with reporter-labeled anti-humanantibody to bind reporter to the reagent in proportion to the amount ofbound anti-antigen antibody on the solid support. The reagent is againwashed to remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or calorimetric substrate(Sigma, St. Louis, Mo.).

[0222] The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

[0223] Thus, the invention provides an assay system or kit for carryingout this diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

[0224] Therapeutic and Diagnostic Uses

[0225] The present invention is also directed, in part, to human TIMP-4which has, as a defining characteristic, the ability to inhibit theaction of MMP's. The human TIMP-4 polypeptide may be employed as ametalloproteinase inhibitor to prevent tumor invasion and angiogenesesand subsequent metastases. The human TIMP-4 polypeptide may also beemployed to treat arthritic diseases, such as rheumatoid arthritis andosteoarthritis, soft tissue rheumatism, polychondritis and tendonitis;and bone resorption diseases, such as osteoporosis, Paget's disease,hyperparathyroidism and cholesteatoma. Human TIMP-4 may also be employedto prevent enhanced collagen destruction that occurs in association withdiabetes, the recessive classes of dystrophic epidermolysis bullosa,periodontal disease and related consequences of gingival production ofcollagenase. human TIMP-4 may also be employed to inhibit PMNLcollagenase release following cellular infiltration to inflamed gingiva,including combatting the greater susceptibility of diabetes patients toperiodontal disease.

[0226] Human TIMP-4 may also be employed to treat corneal ulceration,for example, that induced by alkali or other burns, by radiation, byVitamin E or retinoid deficiency; ulceration of the skin andgastrointestinal tract, and abnormal wound healing, and post-operativeconditions including colonic anastomosis, in which collagenase levelsare raised.

[0227] MMP's mediate tumor growth in situ. Accordingly, human TIMP-4 maybe used to block the destruction of cellular basement membranes, whichis the mechanism by which cancer cells metastasize. MMP's have beenimplicated in neovascularization required to support tumor growth andsurvival, in the tissue remodeling required to accommodate the growingprimary and secondary tumors, and in the penetration of tumor cellsthrough the basement membranes of the vascular walls during metastasis.

[0228] Thus, in specific embodiments, the invention provides fortreatment or prevention of various diseases and disorders involving cellproliferation, tumor cell invasion, and tumor angiogenesis.

[0229] TIMP-4 polynucleotides, polypeptides, antibodies (e.g., agonisticanti-TIMP-4 antibodies) and/or agonists of the invention, may be usedfor therapeutic/prophylactic purposes alone or in combination with othertherapeutics useful in the treatment of cancer and hyperproliferative ordysproliferative disorders. Diseases and disorders involving celloverproliferation are treated or prevented by administration of a TIMP-4polynucleotide, polypeptide, antibody and/or agonist of the inventionthat promotes (i.e., increases or supplies) TIMP-4 function.

[0230] Diseases and disorders involving cell overproliferation that canbe treated or prevented using the polynucleotides, polypeptides,antibodies, and/or agonists of the invention include, but are notlimited to, malignancies, premalignant conditions (e.g., hyperplasia,metaplasia, dysplasia), benign tumors, hyperproliferative disorders,benign dysproliferative disorders, etc.

[0231] Malignancies and related disorders that can be treated orprevented by administration of a TIMP-4 polynucleotide, polypeptide,antibody and/or agonist of the invention, include but are not limitedto, leukemia, acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, myeloblastic leukemia, promyelocytic leukemia,myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronicleukemia, chronic myelocytic (granulocytic) leukemia, chroniclymphocytic leukemia, Polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's disease, multiple myeloma, Waldenstrom'smacroglobulinemia, Heavy chain disease, solid tumors, sarcomas andcarcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, colorectal carcinoma, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, uterine cancer, testicular tumor, lung carcinoma, small celllung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, andesophageal carcinoma.(for a review of such disorders, see Fishman etal., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia).

[0232] In a specific embodiment, digestive tract tumors are treated orprevented, including but not limited to, esophageal, stomach, colon, andcolorectal cancers. In another specific embodiment, airway cancers suchas lung cancers (e.g., small cell lung carcinoma) and nasopharyngealcarcinoma are treated or prevented. In yet other specific embodiments,malignancy or dysproliferative changes (such as metaplasias anddysplasias), or hyperproliferative disorders, are treated or preventedin the head, neck, cervix, kidney, stomach, skin, ovary, bladder,breast, colon, lung, or uterus. In other specific embodiments, sarcoma,or leukemia is treated or prevented. In another particular embodiment,osteosarcoma or renal cell carcinoma is treated or prevented.

[0233] TIMP-4 polynucleotides, polypeptides, antibodies and/or agonistsof the invention can also be administered to treat premalignantconditions and to prevent progression to a neoplastic or malignantstate, including but not limited to, those disorders listed above. Suchprophylactic or therapeutic use is indicated in conditions known orsuspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, 1976, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79.)Hyperplasia is a form of controlled cell proliferation involving anincrease in cell number in a tissue or organ, without significantalteration in structure or function. As but one example, endometrialhyperplasia often precedes endometrial cancer. Metaplasia is a form ofcontrolled cell growth in which one type of adult or fullydifferentiated cell substitutes for another type of adult cell.Metaplasia can occur in epithelial or connective tissue cells. Atypicalmetaplasia involves a somewhat disorderly metaplastic epithelium.Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia; it is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplastic cells often haveabnormally large, deeply stained nuclei, and exhibit pleomorphism.Dysplasia characteristically occurs where there exists chronicirritation or inflammation, and is often found in the cervix,respiratory passages, oral cavity, and gall bladder.

[0234] In a specific embodiment, a TIMP-4 polypeptide, polynucleotide,antibody, and/or agonist of the invention is administered to a humanpatient to prevent progression to breast, colon, lung, stomach oruterine cancer, or melanoma or sarcoma.

[0235] In another embodiment of the invention, a polynucleotide,polypeptide, antibody and/or agonist of the invention is used to treator prevent hyperproliferative or benign dysproliferative disorders.Specific embodiments are directed to treatment or prevention of benigntumors, fibrocystic conditions, and tissue hypertrophy (e.g., prostatichyperplasia).

[0236] In a specific embodiment TIMP-4 polynucleotides, polypeptides,antibodies, and/or agonists of the invention are administered to treator prevent an immune system related disease, disorder or condition. In apreferred embodiment, the immune system disease, disorder, or conditionis an autoimmune diesease, disorder, or condition. In a most preferredembodiment, the immune system diesease, disorder, or condition, isrheumatoid arthritis.

[0237] MMP's are responsible for localized degradation of the follicularwall during ovulation and localized degradation of the uterine wall forblastocyte implantation. Accordingly, human TIMP-4 may be employed as acontraceptive.

[0238] Human TIMP-4 may also be employed as a general growth factor totreat restenosis and similar diseases. Human TIMP-4 may be employedparticularly as a growth factor for erythroid cell lineages.

[0239] TIMP-4 is a strong inhibitor of metalloproteinases which degradestructural components of tissues and are involved in the remodeling oftissues in normal and certain pathological states. Accordingly,potential therapeutic applications of the TIMP-4 polynucleotides,polypeptides, antibodies, and/or agonists of the invention include, butare not limited to, the treatment and/or prevention of restenosis, orobstruction of blood vessels, such as coronary arteries, and heartfailure. Restenosis is a medical condition characterized by theconstriction of coronary arteries. Restenosis usually occurs followingtreatment to open coronary arteries clogged by plaque accumulation.Balloon angioplasty, insertion of a catheter into the clogged arteryfollowed by expansion of a balloon at the site of blockage, compressesthe plaque and opens the arteries. This procedure can damage the wall ofthe artery. The damaged vessel often responds to the balloon angioplastyinjury by overgrowth, which can lead to reconstriction of the artery.While not intending to be bound by theory, it is believed that TIMP-4acts to treat or prevent restenosis by blocking metalloproteinases, afamily of genes that become active after injury to the artery and arethought to play a major role in restenosis.

[0240] TIMP-4 polynucleotides or polypeptides, or agonists orantagonists of TIMP-4, encoding TIMP-4 may be used to treat, prevent,and/or diagnose cardiovascular diseases, disorders, and/or conditions,including peripheral artery disease, such as limb ischemia.

[0241] Cardiovascular diseases, disorders, and/or conditions that may betreated, prevented and/or diagnosed with the polynucleotides,polypeptides (including antibodies), agonists and/or antagonists of theinvention include, but are not limited to, cardiovascular abnormalities,such as arterio-arterial fistula, arteriovenous fistula, cerebralarteriovenous malformations, congenital heart defects, pulmonaryatresia, and Scimitar Syndrome. Congenital heart defects include aorticcoarctation, cor triatriatum, coronary vessel anomalies, crisscrossheart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,Eisenmenger complex, hypoplastic left heart syndrome, levocardia,tetralogy of fallot, transposition of great vessels, double outlet rightventricle, tricuspid atresia, persistent truncus arteriosus, and heartseptal defects, such as aortopulmonary septal defect, endocardialcushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricularheart septal defects.

[0242] Cardiovascular diseases, disorders, and/or conditions that may betreated, prevented and/or diagnosed with the polynucleotides,polypeptides (including antibodies), agonists and/or antagonists of theinvention also include, but are not limited to, heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

[0243] Arrhythmias that may be treated, prevented and/or diagnosed withthe polynucleotides, polypeptides (including antibodies), agonistsand/or antagonists of the invention include, but are not lmited to,sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia,extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrialblock, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome,Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome,sick sinus syndrome, tachycardias, and ventricular fibrillation.Tachycardias include paroxysmal tachycardia, supraventriculartachycardia, accelerated idioventricular rhythm, atrioventricular nodalreentry tachycardia, ectopic atrial tachycardia, ectopic junctionaltachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia,Torsades de Pointes, and ventricular tachycardia.

[0244] Heart valve disease that may be treated, prevented and/ordiagnosed with the polynucleotides, polypeptides (including antibodies),agonists and/or antagonists of the invention include, but are notlimited to, aortic valve insufficiency, aortic valve stenosis, hearmurmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valveprolapse, mitral valve insufficiency, mitral valve stenosis, pulmonaryatresia, pulmonary valve insufficiency, pulmonary valve stenosis,tricuspid atresia, tricuspid valve insufficiency, and tricuspid valvestenosis.

[0245] Myocardial diseases that may be treated, prevented and/ordiagnosed with the polynucleotides, polypeptides (including antibodies),agonists and/or antagonists of the invention include, but are notlimited to, alcoholic cardiomyopathy, congestive cardiomyopathy,hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonarysubvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy,endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome,myocardial reperfusion injury, and myocarditis.

[0246] Myocardial ischemias that may be treated, prevented and/ordiagnosed with the polynucleotides, polypeptides (including antibodies),agonists and/or antagonists of the invention include, but are notlimited to, coronary disease, such as angina pectoris, coronaryaneurysm, coronary arteriosclerosis, coronary thrombosis, coronaryvasospasm, myocardial infarction and myocardial stunning.

[0247] Cardiovascular diseases that may be treated, prevented and/ordiagnosed with the polynucleotides, polypeptides (including antibodies),agonists and/or antagonists of the invention also include, but are notlimited to, vascular diseases such as aneurysms, angiodysplasia,angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease,Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneuroticedema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche'sSyndrome, arterial occlusive diseases, arteritis, enarteritis,polyarteritis nodosa, cerebrovascular diseases, disorders, and/orconditions, diabetic angiopathies, diabetic retinopathy, embolisms,thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusivedisease, hypertension, hypotension, ischemia, peripheral vasculardiseases, phlebitis, pulmonary veno-occlusive disease, Raynaud'sdisease, CREST syndrome, retinal vein occlusion, Scimitar syndrome,superior vena cava syndrome, telangiectasia, atacia telangiectasia,hereditary hemorrhagic telangiectasia, varicocele, varicose veins,varicose ulcer, vasculitis, and venous insufficiency.

[0248] Aneurysms that may be treated, prevented and/or diagnosed withthe polynucleotides, polypeptides (including antibodies), agonistsand/or antagonists of the invention include, but are not limited to,dissecting aneurysms, false aneurysms, infected aneurysms, rupturedaneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms,heart aneurysms, and iliac aneurysms.

[0249] Arterial occlusive diseases that may be treated, prevented and/ordiagnosed with the polynucleotides, polypeptides (including antibodies),agonists and/or antagonists of the invention include, but are notlimited to, arteriosclerosis, intermittent claudication, carotidstenosis, fibromuscular dysplasias, mesenteric vascular occlusion,Moyamoya disease, renal artery obstruction, retinal artery occlusion,and thromboangiitis obliterans.

[0250] Cerebrovascular diseases, disorders, and/or conditions that maybe treated, prevented and/or diagnosed with the polynucleotides,polypeptides (including antibodies), agonists and/or antagonists of theinvention include, but are not limited to, carotid artery diseases,cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebralartery diseases, cerebral embolism and thrombosis, carotid arterythrombosis, sinus thrombosis, Wallenberg's syndrome, cerebralhemorrhage, epidural hematoma, subdural hematoma, subaraxhnoidhemorrhage, cerebral infarction, cerebral ischemia (includingtransient), subclavian steal syndrome, periventricular leukomalacia,vascular headache, cluster headache, migraine, and vertebrobasilarinsufficiency.

[0251] Embolisms that may be treated, prevented and/or diagnosed withthe polynucleotides, polypeptides (including antibodies), agonistsand/or antagonists of the invention include, but are not limited to, airembolisms, amniotic fluid embolisms, cholesterol embolisms, blue toesyndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms.Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinalvein occlusion, carotid artery thrombosis, sinus thrombosis,Wallenberg's syndrome, and thrombophlebitis.

[0252] Ischemia that may be treated, prevented and/or diagnosed with thepolynucleotides, polypeptides (including antibodies), agonists and/orantagonists of the invention includes, but are not limited to, cerebralischemia, ischemic colitis, compartment syndromes, anterior compartmentsyndrome, myocardial ischemia, reperfusion injuries, and peripheral limbischemia. Vasculitis includes aortitis, arteritis, Behcet's Syndrome,Churg-Strauss Syndrome, mucocutaneous lymph node syndrome,thromboangiitis obliterans, hypersensitivity vasculitis,Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener'sgranulomatosis.

[0253] TIMP-4 polypeptides may be administered using any method known inthe art, including, but not limited to, gene therapy (e.g., viatechniques known in the art utilizing adenovirus vectors), gene gun,direct needle injection at the delivery site, intravenous injection,topical administration, catheter infusion, biolistic injectors, particleaccelerators, gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps, oral or suppositorial solid pharmaceuticalformulations, decanting or topical applications during surgery, aerosoldelivery. Such methods are known in the art. TIMP-4 polypeptides may beadministered as part of a Therapeutic, described in more detail below.Methods of delivering TIMP-4 polynucleotides are described in moredetail herein.

[0254] The naturally occurring balance between endogenous stimulatorsand inhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-447 (1987).

[0255] The present invention provides for treatment or prevention ofdiseases or disorders associated with neovascularization byadministration of the polynucleotides, polypeptides, antibodies, and/oragonists of the invention. Malignant and metastatic conditions which canbe treated with the polynucleotides, polypeptides, antibodies, and/oragonists of the invention include, but are not limited to, malignancies,solid tumors, and cancers described herein and otherwise known in theart (for a review of such disorders, see Fishman et al., Medicine, 2dEd., J. B. Lippincott Co., Philadelphia (1985)).

[0256] Ocular disorders associated with neovascularization which can betreated or prevented with the TIMP-4 polynucleotides, polypeptides,antibodies, and/or agonists of the present invention include, but arenot limited to: neovascular glaucoma, diabetic retinopathy,retinoblastoma, retrolental fibroplasia, uveitis, retinopathy ofprematurity macular degeneration, corneal graft neovascularization, aswell as other eye inflammatory diseases, ocular tumors and diseasesassociated with choroidal or iris neovascularization. See, e.g., reviewsby Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al.,Surv. Ophthal. 22:291-312 (1978).

[0257] In another embodiment, TIMP-4 polypeptides, polynucleotides,antibodies and/or agonists or antagonists of the invention are used tostimulate differentiation and/or survival of photoreceptor cells and/orto treat or prevent diseases, disorders, or conditions associated withdecreased number, differentiation and/or survival of photoreceptorcells.

[0258] Additionally, disorders which can be treated with the TIMP-4polynucleotides, polypeptides, antibodies, agonists and/or antagonist ofthe present invention include, but are not limited to, hemangioma,arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayedwound healing, granulations, hemophilic joints, hypertrophic scars,nonunion fractures, Osler-Weber syndrome, pyogenic granuloma,scleroderma, trachoma, and vascular adhesions.

[0259] In further embodiments, the TIMP-4 polynucleotides, polypeptides,antibodies, agonists, and/or antagonists of the inveniton, are used topromote wound healing.

[0260] In alternative embodiments, TIMP-4 polynucleotides, polypeptides,antibodies (e.g., antagonistic anti-TIMP-4 antibodies) and/orantagonists of the invention, are useful in the treatment of disordersin which stimulation of new blood vessel development would amelioratethe disorder. Such disorders include, but are not limited to, heartfailure, angina, blood vessel (e.g. coronary artery) blockage andischemia, including critical limb ischemia and refractory myocardialischemia. Antagonistic TIMP-4 polynucleotides of the invention can bedelivered to individuals to using gene therapy techniques and materialsdescribed herein or otherwise known in the art.

[0261] As a result of the ability to stimulate vascular endothelial cellgrowth, TIMP-4 antagonists of the invention, such as for example,antagonistic anti-TIMP-4 antibodies, may be employed in treatment forstimulating re-vascularization of ischemic tissues due to variousdisease conditions such as thrombosis, arteriosclerosis, and othercardiovascular conditions. The polypeptides, polynucleotides,antibodies, agonists and/or antagonists of the present invention mayalso be employed to stimulate angiogenesis and limb regeneration, asdiscussed herein.

[0262] TIMP-4 polynucleotides, polypeptides, antibodies (e.g., agonisticanti-TIMP-4 antibodies), and/or agonists can be used to inhibit MMPmediated extracellular matrix degradation or alternativelydifferentiate, proliferate, and attract cells, and thereby lead to theregeneration of tissues. (See, Science 276:59-87 (1997).) Theregeneration of tissues could be used to repair, replace, or protecttissue damaged by congenital defects, trauma (wounds, bums, incisions,or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

[0263] Tissues that could be regenerated using the present inventioninclude organs (e.g., pancreas, liver, heart, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac), vasculature(including vascular and lymphatics), nervous, hematopoietic, andskeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,regeneration occurs without or decreased scarring. Regeneration also mayinclude angiogenesis.

[0264] Moreover, TIMP-4 polynucleotides, polypeptides, antibodies, andagonists or antagonists of the invention may increase regeneration oftissues difficult to heal. For example, increased tendon/ligamentregeneration would quicken recovery time after damage. TIMP-4polynucleotides, polypeptides, antibodies, and agonists or antagonistsof the present invention could also be used prophylactically in aneffort to avoid damage. Specific diseases that could be treated orprevented include of tendinitis, carpal tunnel syndrome, and othertendon or ligament defects. A further example of tissue regeneration ofnon-healing wounds includes pressure ulcers, ulcers associated withvascular insufficiency, surgical, and traumatic wounds.

[0265] Similarly, nerve and brain tissue could also be regeneratedaccording to the present invention by using TIMP-4 polynucleotides,polypeptides, antibodies, agonists and/or antagonists to, for example,proliferate and differentiate nerve cells. Diseases that could betreated or prevented using this method include, but are not limited to,central and peripheral nervous system diseases, neuropathies, ormechanical and traumatic disorders (e.g., spinal cord disorders, headtrauma, cerebrovascular disease, and stoke). Specifically, diseasesassociated with peripheral nerve injuries, peripheral neuropathy (e.g.,resulting from chemotherapy or other medical therapies), localizedneuropathies, and central nervous system diseases (e.g., Alzheimer'sdisease, Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome), could all be treated using theTIMP-4 polynucleotides, polypeptides, antibodies, and agonists orantagonists of the invention.

[0266] Among the other diseases which TIMP-4 may be employed to treatincludes alveolitis, asthma, psoriasis, glomerulosclerosis, and septicshock since MMP's are involved in the tissue invasiveness of someparasites.

[0267] An effective amount of the TIMP-4 polynucleotides, polypeptides,antibodies and/or agonists or antagonists can be administered in vitro,ex vivo, or in vivo using techniques and compositions described hereindescribed herein (e.g., in the section entitled Therapeutic/ProphylacticAdministration and Composition) or otherwise known in the art. Byadministration of an “effective amount” of TIMP-4 polynucleotides,polypeptides, antibodies and/or agonists or antagonists is intended anamount of the compound that is sufficient to enhance or inhibit acellular response to one or more metalloproteinases. In particular, byadministration of an “effective amount” of an agonist or antagonists isintended an amount effective to enhance or inhibit TIMP-4polynucleotides, polypeptides, antibodies and/or agonists or antagonistsmediated metalloproteinase actiivity. One of ordinary skill willappreciate that effective amounts of an agonist or antagonist can bedetermined empirically and may be employed in pure form or inpharmaceutically acceptable salt, ester or pro-drug form. The agonist orantagonist may be administered in compositions in combination with oneor more pharmaceutically acceptable excipients.

[0268] It will be understood that, when administered to a human patient,the total daily usage of the compounds and compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgement. The specific therapeutically effective doselevel for any particular patient will depend upon factors well known inthe medical arts.

[0269] As a general proposition, the total pharmaceutically effectiveamount of a TIMP-4 polypeptide administered parenterally per dose willbe in the range of about 1 μg/kg/day to 10 mg/kg/day of patient bodyweight, although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the TIMP-4 polypeptides are typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed.

[0270] Pharmaceutical compositions containing the TIMP-4 polypeptides ofthe invention may be administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, drops or transdermal patch), bucally, or as an oralor nasal spray. By “pharmaceutically acceptable carrier” is meant anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrastemal, subcutaneous andintraarticular injection and infusion.

[0271] The pharmaceutical compositions may be administered in aconvenient manner such as by the topical, intravenous, intra-articular,intra-tumor, intraperitoneal, intramuscular, subcutaneous, intranasal orintradermal routes. The pharmaceutical compositions are administered inan amount which is effective for treating and/or prophylaxis of thespecific indication. In general, the pharmaceutical compositions areadministered in an amount of at least about 10 micrograms/kg body weightand in most cases they will be administered in an amount not in excessof about 8 mg/Kg body weight per day and preferably the dosage is fromabout 10 micrograms/kg to about 1 mg/kg body weight daily, taking intoaccount the routes of administration, symptoms, etc.

[0272] The compositions of the invention may be administered alone or incombination with other therapeutic agents, including but not limited to,chemotherapeutic agents, anti-angiogenic agents, angiogenic agents,anti-opportunistic infection agents, antivirals, antibiotics, steroidaland non-steroidal anti-inflammatories, immunosuppressants, conventionalimmunotherapeutic agents and cytokines. Combinations may be administeredeither concomitantly, e.g., as an admixture, separately butsimultaneously or concurrently; or sequentially. This includespresentations in which the combined agents are administered together asa therapeutic mixture, and also procedures in which the combined agentsare administered separately but simultaneously, e.g., as throughseparate intravenous lines into the same individual. Administration “incombination” further includes the separate administration of one of thecompounds or agents given first, followed by the second.

[0273] In one embodiment, the compositions of the invention areadministered in combination with a member of the TNF family. TNF,TNF-related or TNF-like molecules that may be administered with thecompositions of the invention include, but are not limited to, solubleforms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known asTNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL,CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (InternationalPublication No. WO 96/14328), AIM-I (International Publication No. WO97/33899), AIM-II (International Publication No. WO 97/34911), APRIL,endokine-alpha (International Publication No. WO 98/07880), TR6(International Publication No. WO 98/30694), OPG, and neutrokine-alpha(International application publication number WO 98/18921), TWEAK, OX40,and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27,CD40 and 4-IBB, TR2 (International application publication number WO96/34095), DR3 (International Publication No. WO 97/33904), DR4(International application publication number WO 98/32856), TR5(International application publication number WO 98/30693), TR7(International application publication number WO 98/41629), TRANK, TR9(International application publication number WO 98/56892), TRIO(International application publication number WO 98/54202),312C2(International application publication number WO 98/06842), and TR12.

[0274] Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

[0275] In specific embodiments, compositions of the invention areadministered in combination with immunosuppressants. Immunosuppressantspreparations that may be administered with the compositions of theinvention include, but are not limited to, ORTHOCLONE™ (OKT3),SANDIMMUNE™/NEORAL™/SANGDYA™ (cyclosporin), PROGRAF™ (tacrolimus),CELLCEPT™ (mycophenolate), Azathioprine, glucorticosteroids, andRAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants maybe used to prevent rejection of organ or bone marrow transplantation.

[0276] In certain embodiments, compositions of the invention areadministered in combination with antiretroviral agents, nucleosidereverse transcriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the compositionsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC),ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith compositions of the invention to treat AIDS and/or to prevent ortreat HIV infection.

[0277] In other embodiments, compositions of the invention may beadministered in combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe compositions of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, compositions of theinvention are used in any combination withTRINETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, compositions of the invention are used in any combinationwith ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ toprophylactically treat or prevent an opportunistic Mycobacterium aviumcomplex infection. In another specific embodiment, compositions of theinvention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™,and/or AZITHROMYCIN™ to prophylactically treat or prevent anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, compositions of the invention are used in any combinationwith GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylacticallytreat or prevent an opportunistic cytomegalovirus infection. In anotherspecific embodiment, compositions of the invention are used in anycombination with FLUCONAZOLE™, ITRFRACONAZOLE™, and/or KETOCONAZOLE™ toprophylactically treat or prevent an opportunistic fungal infection. Inanother specific embodiment, compositions of the invention are used inany combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, compositions of theinvention are used in any combination with PYRIMETHAMINE™ and/orLEUCOVORIN™ to prophylactically treat or prevent an opportunisticToxoplasma gondii infection. In another specific embodiment,compositions of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent anopportunistic bacterial infection.

[0278] In a further embodiment, the compositions of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the compositions of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine

[0279] In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, amoxicillin, aminoglycosides, beta-lactam(glycopeptide), beta-lactamases, Clindamycin, chloramphenicol,cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim,trimethoprim-sulfamthoxazole, and vancomycin.

[0280] In an additional embodiment, the compositions of the inventionare administered alone or in combination with an anti-inflammatoryagent. Anti-inflammatory agents that may be administered with thecompositions of the invention include, but are not limited to,glucocorticoids and the nonsteroidal anti-inflammatories,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

[0281] In another embodiment, compostions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the compositionsof the invention include, but are not limited to, antibiotic derivatives(e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin);antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil,5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid,plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g.,carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophospharnide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0282] In a specific embodiment, compositions of the invention areadministered in combination with CHOP (cyclophosphamide, doxorubicin,vincristine, and prednisone) or any combination of the components ofCHOP. In another embodiment, compositions of the invention areadministered in combination with Rituximab. In a further embodiment,compositions of the invention are administered with Rituxmab and CHOP,or Rituxmab and any combination of the components of CHOP.

[0283] In an additional embodiment, the compositions of the inventionare administered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, GM-CSF, G-CSF, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12,IL13, IL15, anti-CD40, CD40L, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha,and TNF-beta. In another embodiment, compositions of the invention maybe administered with any interleukin, including, but not limited to,IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, and IL-21. In a preferred embodiment, the compositions of theinvention are administered in combination with TNF-alpha. In anotherpreferred embodiment, the compositions of the invention are administeredin combination with IFN-alpha.

[0284] In an additional embodiment, the compositions of the inventionare administered alone or in combination with an anti-angiogenic agent.Anti-angiogenic agents that may be administered with the compositions ofthe invention include, but are not limited to, Angiostatin (Entremed,Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Ma.),anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel(Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, TissueInhibitor of Metalloproteinase-2, VEGI, Plasminogen ActivatorInhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of thelighter “d group” transition metals.

[0285] Lighter “d group” transition metals include, for example,vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species.Such transition metal species may form transition metal complexes.Suitable complexes of the above-mentioned transition metal speciesinclude oxo transition metal complexes.

[0286] Representative examples of vanadium complexes include oxovanadium complexes such as vanadate and vanadyl complexes. Suitablevanadate complexes include metavanadate and orthovanadate complexes suchas, for example, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

[0287] Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

[0288] A wide variety of other anti-angiogenic factors may also beutilized within the context of the present invention. Representativeexamples include, but are not limited to, platelet factor 4; protaminesulphate; sulphated chitin derivatives (prepared from queen crabshells), (Murata et al., Cancer Res. 51:22-26, 1991); SulphatedPolysaccharide Peptidoglycan Complex (SP-PG) (the function of thiscompound may be enhanced by the presence of steroids such as estrogen,and tamoxifen citrate); Staurosporine; modulators of matrix metabolism,including for example, proline analogs, cishydroxyproline,d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl,aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone;Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum;ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992);Chymostatin (Tomkinson et al., Biochem J. 286:475-480, 1992);Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin(Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate(“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987);anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem.262(4):1659-1664, 1987); Bisantrene (National Cancer Institute);Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic aciddisodium or “CCA”; (Takeuchi et al., Agents Actions 36:312-316, 1992);and metalloproteinase inhibitors such as BB94.

[0289] Additional anti-angiogenic factors that may also be utilizedwithin the context of the present invention include Thalidomide,(Celgene, Warren, N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J.Folkman J Pediatr. Surg. 28:445-51 (1993)); an integrin alpha v beta 3antagonist (C. Storgard et al., J Clin. Invest. 103:47-54 (1999));carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National CancerInstitute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXiGENE, Boston,Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.);TNP-470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca(London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251(PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin;Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide(Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat(AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex);Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and5-Fluorouracil.

[0290] Anti-angiogenic agents that may be administed in combination withthe compounds of the invention may work through a variety of mechanismsincluding, but not limited to, inhibiting proteolysis of theextracellular matrix, blocking the function of endothelialcell-extracellular matrix adhesion molecules, by antagonizing thefunction of angiogenesis inducers such as growth factors, and inhibitingintegrin receptors expressed on proliferating endothelial cells.Examples of anti-angiogenic inhibitors that interfere with extracellularmatrix proteolysis and which may be administered in combination with thecompositons of the invention include, but are not lmited to, AG-3340(Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.),BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-27032A(Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford,UK), and Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenicinhibitors that act by blocking the function of endothelialcell-extracellular matrix adhesion molecules and which may beadministered in combination with the compositons of the inventioninclude, but are not lmited to, EMD-121974 (Merck KcgaA Darmstadt,Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune, Gaithersburg,Md.). Examples of anti-angiogenic agents that act by directlyantagonizing or inhibiting angiogenesis inducers and which may beadministered in combination with the compositons of the inventioninclude, but are not Imited to, Angiozyme (Ribozyme, Boulder, Colo.),Anti-VEGF antibody (Genentech, S. San Francisco, Calif.),PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. SanFrancisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, N.J.),and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectlyinhibit angiogenesis. Examples of indirect inhibitors of angiogenesiswhich may be administered in combination with the compositons of theinvention include, but are not Imited to, IM-862 (Cytran, Kirkland,Wash.), Interferon-alpha, IL-12 (Roche, Nutley, N.J.), and Pentosanpolysulfate (Georgetown University, Washington, D.C.).

[0291] In particular embodiments, the use of compositions of theinvention in combination with anti-angiogenic agents is contemplated forthe treatment, prevention, and/or amelioration of an autoimmune disease,such as for example, an autoimmune disease described herein.

[0292] In a particular embodiment, the use of compositions of theinvention in combination with anti-angiogenic agents is contemplated forthe treatment, prevention, and/or amelioration of arthritis. In a moreparticular embodiment, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of rheumatoid arthritis.

[0293] In an additional embodiment, the compositions of the inventionare administered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the compositions of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PlGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B-186 (VEGF-B186), as disclosed in InternationalPublication Number WO 96/26736; Vascular Endothelial Growth Factor-D(VEGF-D), as disclosed in International Publication Number WO 98/02543;Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/07832; and Vascular EndothelialGrowth Factor-E (VEGF-E), as disclosed in German Patent NumberDE19639601. The above mentioned references are incorporated herein byreference herein.

[0294] In an additional embodiment, the compositions of the inventionare administered in combination with Fibroblast Growth Factors.Fibroblast Growth Factors that may be administered with the compositionsof the invention include, but are not limited to, FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12,FGF-13, FGF-14, and FGF-15.

[0295] In additional embodiments, the compositions of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

[0296] This invention is also related to the use of the human TIMP-4gene as part of a diagnostic assay for detecting diseases orsusceptibility to diseases related to the presence of mutated humanTIMP-4.

[0297] Individuals carrying mutations in the human TIMP-4 gene may bedetected at the DNA level by a variety of techniques. Nucleic acids fordiagnosis may be obtained from a patient's cells, such as from blood,urine, saliva, tissue biopsy and autopsy material. The genomic DNA maybe used directly for detection or may be amplified enzymatically byusing PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.RNA or cDNA may also be used for the same purpose. As an example, PCRprimers complementary to the nucleic acid encoding human TIMP-4 can beused to identify and analyze human TIMP-4 mutations. For example,deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to radiolabeled humanTIMP-4 RNA or alternatively, radiolabeled human TIMP-4 antisense DNAsequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

[0298] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

[0299] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0300] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0301] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0302] The present invention also relates to a diagnostic assay fordetecting altered levels of human TIMP-4 protein in various tissuessince an over-expression of the proteins compared to normal controltissue samples may detect the presence of a disease or susceptibility toa disease regulated by human TIMP-4. Assays used to detect levels ofhuman TIMP-4 protein in a sample derived from a host are well-known tothose of skill in the art and include radioimmunoassays,competitive-binding assays, Western Blot analysis, ELISA assays and“sandwich” assay. An ELISA assay (Coligan, et al., Current Protocols inImmunology, 1(2), Chapter 6, (1991)) initially comprises preparing anantibody specific to the human TIMP-4 antigen, preferably a monoclonalantibody. In addition a reporter antibody is prepared against themonoclonal antibody. To the reporter antibody is attached a detectablereagent such as radioactivity, fluorescence or, in this example, ahorseradish peroxidase enzyme. A sample is removed from a host andincubated on a solid support, e.g. a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein like BSA. Next,the monoclonal antibody is incubated in the dish during which time themonoclonal antibodies attach to any human TIMP-4 proteins attached tothe polystyrene dish. All unbound monoclonal antibody is washed out withbuffer. The reporter antibody linked to horseradish peroxidase is nowplaced in the dish resulting in binding of the reporter antibody to anymonoclonal antibody bound to human TIMP-4. Unattached reporter antibodyis then washed out. Peroxidase substrates are then added to the dish andthe amount of color developed in a given time period is a measurement ofthe amount of human TIMP-4 protein present in a given volume of patientsample when compared against a standard curve.

[0303] A competition assay may be employed wherein antibodies specificto human TIMP-4 are attached to a solid support and labeled human TIMP-4and a sample derived from the host are passed over the solid support andthe amount of label detected, for example by liquid scintillationchromatography, can be correlated to a quantity of human TIMP-4 in thesample.

[0304] A “sandwich” assay is similar to an ELISA assay. In a “sandwich”assay human TIMP-4 is passed over a solid support and binds to antibodyattached to a solid support. A second antibody is then bound to thehuman TIMP-4. A third antibody which is labeled and specific to thesecond antibody is then passed over the solid support and binds to thesecond antibody and an amount can then be quantitated.

[0305] This invention also provides a method of screening compounds toidentify those which are agonists or antagonists to be human TIMP-4polypeptide. An example of such a method comprises obtaining mammaliantissue comprising an extra-cellular matrix, for example, bovineradiocarpal joints. The articular cartilage is cut into smaller disksand labeled with ³⁵S-sodium sulfate (10 micro Ci/ml) in DMEM for asufficient time for the cartilage to incorporate the labeled Sodiumsulfate. An MMP, for example, stromelysin, or IL1 or TNF is then addedto the cartilage disks under appropriate conditions such that tissuebreakdown would normally occur. Human TIMP-4 and the compounds to bescreened are then added to the reaction mixture for a sufficient timefor the MMP to normally break down the cartilage disks. The supernatant,which is the media outside the cartilage disks, is then collected andradioactivity is counted by a liquid scintillation counter. Thepercentage of ³⁵S released into the media is then calculated. Thisrelease of ³⁵S-GAG is representative of the proteoglycan pool in theextracellular matrix of cartilage, and reflects proteoglycan degradationby the MMP. The amount of ³⁵S-GAG, as determined by liquid scintillationchromatography, is then compared to a control assay done in the absenceof the compound to be screened and the ability of the compound toagonize or antagonize the action of human TIMP-4 may then be determined.

[0306] Examples of potential human TIMP-4 antagonists, in addition tothose identified above, include an antibody, or in some cases, anoligonucleotide, which binds to the polypeptide. Alternatively, apotential antagonist may be a mutated form of human TIMP-4, whichrecognizes natural substrates, but is inactive, and thereby prevent theaction of human TIMP-4.

[0307] Potential human TIMP-4 antagonists also include antisenseconstructs prepared using antisense technology. Antisense technology canbe used to control gene expression through triple-helix formation orantisense DNA or RNA, both of which methods are based on binding of apolynucleotide to DNA or RNA. For example, the 5′ coding portion of thepolynucleotide sequence, which encodes for the mature polypeptides ofthe present invention, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix -see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervanet al., Science, 251: 1360 (1991)), thereby preventing transcription andthe production of human TIMP-4. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the human TIMP-4 (antisense—Okano, J. Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988)). The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of humanTIMP-4.

[0308] Another potential human TIMP-4 antagonist is a small moleculewhich binds to and occupies the active site of the human TIMP-4 therebypreventing human TIMP-4 from interacting with MMP's such that normalbiological activity is prevented. Examples of small molecules includebut are not limited to small peptides or peptide-like molecules, forexample a peptide-bonded molecule.

[0309] The human TIMP-4 antagonists may be employed for tissue repairand remodeling, for example, where destruction of scar tissue isdesired. In some situations, enhanced connective tissue turnover orremodeling may be desirable, e.g. in resorption of scar tissue; inuterine involution post-partum; in remodeling of fibrotic deposits inthe lung, liver or joints. To appropriately control turnover ofextra-cellular matrix proteins in these situations would require abalance between the MMP's and human TIMP-4 to appropriately controldegradation.

[0310] The polypeptides and agonists or antagonists that are alsopolypeptides may be employed in accordance with the present invention byexpression of such polypeptides in vivo, which is often referred to as“gene therapy.”

[0311] Gene Therapy

[0312] The invention also encompasses gene therapy methods for treatingor preventing disorders, diseases and conditions, such as, for examplerestenosis. Vectors and techniques described herein (e.g, below or inthe Antibody section of the application) or known in the art may beroutinely applied or modified for such therapy. Gene therapy methodsrelate to the introduction of nucleic acid (DNA, RNA and antisense DNAor RNA) sequence of the invention into an animal to achieve expressionof the TIMP-4 polypeptide of the present invention. This method requiresa polynucleotide which codes for a TIMP-4 polypeptide operatively linkedto a promoter and any other genetic elements necessary for theexpression of the polypeptide by the target tissue. Such gene therapyand delivery techniques are known in the art, see, for example,WO90/11092, which is herein incorporated by reference.

[0313] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to aTIMP-4 polynucleotide ex vivo, with the engineered cells then beingprovided to a patient to be treated with the polypeptide. Such methodsare well-known in the art. For example, see Belldegrun, A., et al., J.Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., CancerResearch 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato,L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al.,Gene Therapy 4:1246-1255 (1997); and Zhang, J. -F. et al., Cancer GeneTherapy 3: 31-38 (1996)), which are herein incorporated by reference. Inone embodiment, the cells which are engineered are arterial cells. Thearterial cells may be reintroduced into the patient through directinjection to the artery, the tissues surrounding the artery, or throughcatheter injection.

[0314] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5:3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT PublicationWO94/12649; and Wang, et al., 1995, Gene Therapy 2:775-783.

[0315] As discussed in more detail herein (e.g., below and in theantibody section of the application), the TIMP-4 polynucleotideconstructs can be delivered by any method that delivers injectablematerials to the cells of an animal, such as, injection into theinterstitial space of tissues (heart, muscle, skin, lung, liver, and thelike). The TIMP-4 polynucleotide constructs may be delivered in apharmaceutically acceptable liquid or aqueous carrier.

[0316] In one embodiment, the TIMP-4 polynucleotide is delivered as anaked polynucleotide. The term “naked” polynucleotide, DNA or RNA refersto sequences that are free from any delivery vehicle that acts toassist, promote or facilitate entry into the cell, including viralsequences, viral particles, liposome formulations, lipofectin orprecipitating agents and the like. However, the TIMP-4 polynucleotidescan also be delivered in liposome formulations and lipofectinformulations and the like can be prepared by methods well known to thoseskilled in the art. Such methods are described, for example, in U.S.Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are hereinincorporated by reference.

[0317] The TIMP-4 polynucleotide vector constructs used in the genetherapy method are preferably constructs that will not integrate intothe host genome nor will they contain sequences that allow forreplication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

[0318] Any strong promoter known to those skilled in the art can be usedfor driving the expression of TIMP-4 polynucleotide sequence. Suitablepromoters include adenoviral promoters, such as the adenoviral majorlate promoter; or heterologous promoters, such as the cytomegalovirus(CMV) promoter; the respiratory syncytial virus (RSV) promoter;inducible promoters, such as the MMT promoter, the metallothioneinpromoter; heat shock promoters; the albumin promoter; the ApoAIpromoter; human globin promoters; viral thymidine kinase promoters, suchas the Herpes Simplex thymidine kinase promoter; retroviral LTRs; theb-actin promoter; and human growth hormone promoters. The promoter alsomay be the native promoter for TIMP-4.

[0319] Unlike other gene therapy techniques, one major advantage ofintroducing naked nucleic acid sequences into target cells is thetransitory nature of the polynucleotide synthesis in the cells. Studieshave shown that non-replicating DNA sequences can be introduced intocells to provide production of the desired polypeptide for periods of upto six months.

[0320] The TIMP-4 polynucleotide construct can be delivered to theinterstitial space of tissues within the an animal, including of muscle,skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular, fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

[0321] For the naked nucleic acid sequence injection, an effectivedosage amount of DNA or RNA will be in the range of from about 0.05mg/kg body weight to about 50 mg/kg body weight. Preferably the dosagewill be from about 0.005 mg/kg to about 20 mg/kg and more preferablyfrom about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan ofordinary skill will appreciate, this dosage will vary according to thetissue site of injection. The appropriate and effective dosage ofnucleic acid sequence can readily be determined by those of ordinaryskill in the art and may depend on the condition being treated and theroute of administration.

[0322] The preferred route of administration is by the parenteral routeof injection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked TIMP-4 DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

[0323] The naked polynucleotides are delivered by any method known inthe art, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, and so-called “gene guns”. These delivery methods are known inthe art.

[0324] The constructs may also be delivered with delivery vehicles suchas viral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

[0325] In certain embodiments, the TIMP-4 polynucleotide constructs arecomplexed in a liposome preparation. Liposomal preparations for use inthe instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA(1987) 84:7413-7416, which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which isherein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is hereinincorporated by reference), in functional form.

[0326] Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated byreference). Other commercially available liposomes include transfectace(DDAB/DOPE) and DOTAP/DOPE (Boehringer).

[0327] Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

[0328] Similarly, anionic and neutral liposomes are readily available,such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easilyprepared using readily available materials. Such materials includephosphatidyl, choline, cholesterol, phosphatidyl ethanolamine,dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol(DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. Thesematerials can also be mixed with the DOTMA and DOTAP starting materialsin appropriate ratios. Methods for making liposomes using thesematerials are well known in the art.

[0329] For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

[0330] The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology (1983), 101:512-527, which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell (1979) 17:77); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA (1979) 76:145); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75:145;Schaefer-Ridder et al., Science (1982) 215:166), which are hereinincorporated by reference.

[0331] Generally, the ratio of DNA to liposomes will be from about 10:1to about 1:10. Preferably, the ration will be from about 5:1 to about1:5. More preferably, the ration will be about 3:1 to about 1:3. Stillmore preferably, the ratio will be about 1:1.

[0332] U.S. Pat. No. 5,676,954 (which is herein incorporated byreference) reports on the injection of genetic material, complexed withcationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355,4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication no. WO 94/9469 (which areherein incorporated by reference) provide cationic lipids for use intransfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466,5,693,622, 5,580,859, 5,703,055, and international publication no. WO94/9469 (which are herein incorporated by reference) provide methods fordelivering DNA-cationic lipid complexes to mammals.

[0333] In certain embodiments, cells are engineered, ex vivo or in vivo,using a retroviral particle containing RNA which comprises a sequenceencoding TIMP-4. Retroviruses from which the retroviral plasmid vectorsmay be derived include, but are not limited to, Moloney Murine LeukemiaVirus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus,avian leukosis virus, gibbon ape leukemia virus, human immunodeficiencyvirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

[0334] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, Ψ-2, Ψ-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy 1:5-14 (1990), which is incorporated herein by reference in itsentirety. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

[0335] The producer cell line generates infectious retroviral vectorparticles which include polynucleotide encoding TIMP-4. Such retroviralvector particles then may be employed, to transduce eukaryotic cells,either in vitro or in vivo. The transduced eukaryotic cells will expressTIMP-4.

[0336] In certain other embodiments, cells are engineered, ex vivo or invivo, with TIMP-4 polynucleotide contained in an adenovirus vector.Adenovirus can be manipulated such that it encodes and expresses TIMP-4,and at the same time is inactivated in terms of its ability to replicatein a normal lytic viral life cycle. Adenovirus expression is achievedwithout integration of the viral DNA into the host cell chromosome,thereby alleviating concerns about insertional mutagenesis. Furthermore,adenoviruses have been used as live enteric vaccines for many years withan excellent safety profile (Schwartz, A. R. et al. (1974) Am. Rev.Respir. Dis.109:233-238). Finally, adenovirus mediated gene transfer hasbeen demonstrated in a number of instances including transfer ofalpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M.A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell68:143-155). Furthermore, extensive studies to attempt to establishadenovirus as a causative agent in human cancer were uniformly negative(Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

[0337] In cases where an adenovirus is used as an expression vector, theTIMP-4 coding sequence of interest may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingthe TIMP-4 molecule in infected hosts. (e.g., see Logan & Shenk, 1984,Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals mayalso be required for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see Bittner et al., 1987, Methods in Enzymol.153:51-544).

[0338] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.204:289-300; U.S. Pat. No. 5,436,146).

[0339] Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992);Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al.,Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692(1993); U.S. Pat. No. 6,040,174, U.S. Pat. No. 6,013,638, and U.S. Pat.No. 5,652,224, each of which are herein incorporated by reference in itsentirety. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the El region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention. (Seee.g., U.S. Pat. Nos. 6,040,174 and 6,013,638, the contents of each ofwhich are incorporated by reference in its entirety).

[0340] Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

[0341] In certain other embodiments, the cells are engineered, ex vivoor in vivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol.158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377,the contents of each of which are herein incorporated by reference inits entirety.

[0342] For example, an appropriate AAV vector for use in the presentinvention will include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The TIMP-4 polynucleotideconstruct is inserted into the AAV vector using standard cloningmethods, such as those found in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAVvector is then transfected into packaging cells which are infected witha helper virus, using any standard technique, including lipofection,electroporation, calcium phosphate precipitation, etc. Appropriatehelper viruses include adenoviruses, cytomegaloviruses, vacciniaviruses, or herpes viruses. Once the packaging cells are transfected andinfected, they will produce infectious AAV viral particles which containthe TIMP-4 polynucleotide construct. These viral particles are then usedto transduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the TIMP-4 polynucleotide construct integrated intoits genome, and will express TIMP-4.

[0343] Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding TIMP-4) via homologous recombination (see, e.g., U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No.WO 96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not normally expressed in the cells, or isexpressed at a lower level than desired.

[0344] Polynucleotide constructs are made, using standard techniquesknown in the art, which contain the promoter with targeting sequencesflanking the promoter. Suitable promoters are described herein. Thetargeting sequence is sufficiently complementary to an endogenoussequence to permit homologous recombination of the promoter-targetingsequence with the endogenous sequence. The targeting sequence will besufficiently near the 5′ end of the TIMP-4 desired endogenouspolynucleotide sequence so the promoter will be operably linked to theendogenous sequence upon homologous recombination.

[0345] The promoter and the targeting sequences can be amplified usingPCR. Preferably, the amplified promoter contains distinct restrictionenzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

[0346] The promoter-targeting sequence construct is delivered to thecells, either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

[0347] The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous TIMP-4 sequence is placedunder the control of the promoter. The promoter then drives theexpression of the endogenous TIMP-4 sequence.

[0348] The polynucleotides encoding TIMP-4 may be administered alongwith other polynucleotides encoding an angiogenic protein. Examples ofangiogenic proteins include, but are not limited to, acidic and basicfibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growthfactor alpha and beta, platelet-derived endothelial cell growth factor,platelet-derived growth factor, tumor necrosis factor alpha, hepatocytegrowth factor, insulin like growth factor, colony stimulating factor,macrophage colony stimulating factor, granulocyte/macrophage colonystimulating factor, and nitric oxide synthase.

[0349] Preferably, the polynucleotide encoding TIMP-4 contains asecretory signal sequence that facilitates secretion of the protein.Typically, the signal sequence is positioned in the coding region of thepolynucleotide to be expressed towards or at the 5′ end of the codingregion. The signal sequence may be homologous or heterologous to thepolynucleotide of interest and may be homologous or heterologous to thecells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

[0350] Any mode of administration of any of the above-describedpolynucleotides constructs can be used so long as the mode results inthe expression of one or more molecules in an amount sufficient toprovide a therapeutic effect. This includes direct needle injection,systemic injection, catheter infusion, biolistic injectors, particleaccelerators (i.e., “gene guns”), gelfoam sponge depots, othercommercially available depot materials, osmotic pumps (e.g., Alzaminipumps), oral or suppositorial solid (tablet or pill) pharmaceuticalformulations, and decanting or topical applications during surgery. Forexample, direct injection of naked calcium phosphate-precipitatedplasmid into rat liver and rat spleen or a protein-coated plasmid intothe portal vein has resulted in gene expression of the foreign gene inthe rat livers (Kaneda et al., Science 243:375 (1989)).

[0351] A preferred method of local administration is by directinjection. Preferably, a recombinant molecule of the present inventioncomplexed with a delivery vehicle is administered by direct injectioninto or locally within the area of arteries. Administration of acomposition locally within the area of arteries refers to injecting thecomposition centimeters and preferably, millimeters within arteries.

[0352] Another method of local administration is to contact apolynucleotide construct of the present invention in or around asurgical wound. For example, a patient can undergo surgery and thepolynucleotide construct can be coated on the surface of tissue insidethe wound or the construct can be injected into areas of tissue insidethe wound.

[0353] Therapeutic compositions useful in systemic administration,include recombinant molecules of the present invention complexed to atargeted delivery vehicle of the present invention. Suitable deliveryvehicles for use with systemic administration comprise liposomescomprising ligands for targeting the vehicle to a particular site.

[0354] Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

[0355] Determining an effective amount of substance to be delivered candepend upon a number of factors including, for example, the chemicalstructure and biological activity of the substance, the age and weightof the animal, the precise condition requiring treatment and itsseverity, and the route of administration. The frequency of treatmentsdepends upon a number of factors, such as the amount of polynucleotideconstructs administered per dose, as well as the health and history ofthe subject. The precise amount, number of doses, and timing of doseswill be determined by the attending physician or veterinarian.

[0356] Therapeutic compositions of the present invention can beadministered to any animal, preferably to mammals and birds. Preferredmammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle,horses and pigs, with humans being particularly preferred.

[0357] Kits

[0358] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the pharmaceutical compositions may be employed in conjunctionwith other therapeutic compounds.

[0359] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0360] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0361] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0362] Fluorescence in situ hybridization (FISH) of a cDNA clones to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

[0363] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0364] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0365] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0366] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0367] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0368] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0369] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0370] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0371] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0372] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0373] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 microgram of plasmid or DNA fragment is used withabout 2 units of enzyme in about 20 microliters of buffer solution. Forthe purpose of isolating DNA fragments for plasmid construction,typically 5 to 50 micrograms of DNA are digested with 20 to 250 units ofenzyme in a larger volume. Appropriate buffers and substrate amounts forparticular restriction enzymes are specified by the manufacturer.Incubation times of about 1 hour at 37° C. are ordinarily used, but mayvary in accordance with the supplier's instructions. After digestion thereaction is electrophoresed directly on a polyacrylamide gel to isolatethe desired fragment.

[0374] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980).

[0375] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0376] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units to T4 DNAligase (“ligase”) per 0.5 micrograms of approximately equimolar amountsof the DNA fragments to be ligated.

[0377] Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

[0378] The entire disclosure of each document cited (including patents,patent applications, journal articles, abstracts, laboratory manuals,books, or other disclosures) in the Background of the Invention,Detailed Description, and Examples of this specification is herebyincorporated by reference in its entirety.

[0379] In addition, the entire disclosure, including the specificationsand sequence listings, of related U.S. application Ser. No. 09/387,525,filed Sep. 1, 1999; Ser. No. 08/463,261, filed Jun. 5, 1995; No.60/217,419, filed Jul. 11, 2000; No. 60/220,829, filed Jul. 26, 2000;and International Application No. PCT/US94/14498, filed Dec. 13, 1994(in English), are each hereby incorporated by reference in theirentireties.

[0380] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLE 1

[0381] Bacterial Expression and Purification of Human TIMP-4

[0382] The DNA sequence encoding for human TIMP-4, ATCC #75946, isinitially amplified using PCR oligonucleotide primers corresponding tothe 5′ and sequences of the processed human TIMP-4 protein (minus thesignal peptide sequence) and the vector sequences 3′ to the TIMP-4 gene.Additional nucleotides corresponding to human TIMP-4 were added to the5′ and 3′ sequences respectively. The 5′ oligonucleotide primer has thesequence 5′ GCCAGAGGATCCTGCAGCTGCGCCCCGGCGCAC 3′ (SEQ ID NO:3) containsa BamHI restriction enzyme site followed by 21 nucleotides of humanTIMP-4 coding sequence starting from the presumed terminal amino acid ofthe processed protein codon. The 3′ sequence 5°C.GGCTTCTAGAACTAGGGCTGAACGATGTCAAC 3′ (SEQ ID NO:4) contains an XbaIsite and is followed by 18 nucleotides of human TIMP-4. The restrictionenzyme sites correspond to the restriction enzyme sites on the bacterialexpression vector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth,Calif., 91311). pQE-9 encodes antibiotic resistance (Amp^(r)), abacterial origin of replication (ori), an IPTG-regulatable promoteroperator (P/O), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-9 was then digested with BamH1 and XbaI.The amplified sequences were ligated into pQE-9 and were inserted inframe with the sequence encoding for the histidine tag and the RBS. Theligation mixture was then used to transform E. coli strain m15/pREP4available from Qiagen by the procedure described in Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,(1989). m15/pREP4 contains multiple copies of the plasmid pREP4, whichexpresses the lacI repressor and also confers kanamycin resistance(Kan^(r)). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies were selected.Plasmid DNA was isolated and confirmed by restriction analysis.

[0383] Clones containing the desired constructs were grown overnight(ON) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells were grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) was then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells were grownan extra 3 to 4 hours. Cells were then harvested by centrifugation. Thecell pellet was solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized human TIMP-4 was purified fromthis solution by chromatography on a Nickel-Chelate column underconditions that allow for tight binding by proteins containing the 6-Histag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984). HumanTIMP-4 (90% pure) was eluted from the column in 6 molar guanidine HCl pH5.0 and for the purpose of renaturation adjusted to 3 molar guanidineHCl, 100 mM sodium phosphate, 10 mmolar glutathione (reduced) and 2mmolar glutathione (oxidized). After incubation in this solution for 12hours the protein was dialyzed to 10 mmolar sodium phosphate.

EXAMPLE 2

[0384] Expression of Recombinant Human TIMP-4 in COS cells

[0385] The expression of human TIMP-4 HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E.coli replication origin, 4) CMVpromoter followed by a polylinker region, a SV40 intron andpolyadenylation site. A DNA fragment encoding the entire human TIMP-4precursor and a HA tag fused in frame to its 3′ end was cloned into thepolylinker region of the vector, therefore, the recombinant proteinexpression is directed under the CMV promoter. The HA tag correspond toan epitope derived from the influenza hemagglutinin protein aspreviously described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M.Connolly, and R. Lemer, 1984, Cell 37, 767). The fusion of HA tag to thetarget protein allows easy detection of the recombinant protein with anantibody that recognizes the HA epitope.

[0386] The plasmid construction strategy is described as follows:

[0387] The DNA sequence ATCC # 75946, encoding for human TIMP-4 wasconstructed by PCR using two primers: the 5′ primer 5′ GCCAGAGGATCCGCCACCATGCCTGGGAGCCCTCGGCCC 3′ (SEQ ID NO:5) contains a BamHI sitefollowed by 21 nucleotides of human TIMP-4 coding sequence starting fromthe initiation codon; the 3′ sequence 5′CGGCTTCTAGAATCAAGCGTAGTCTGGGACGTCG TATGGGTAGGGCTGAACGATGTCAAC 3′ (SEQ IDNO:6) contains complementary sequences to an XbaI site, translation stopcodon, HA tag and the last 18 nucleotides of the human TIMP-4 codingsequence (not including the stop codon). Therefore, the PCR productcontains a BamHI site, human TIMP-4 coding sequence followed by HA tagfused in frame, a translation termination stop codon next to the HA tag,and an XbaI site. The PCR amplified DNA fragment and the vector,pcDNAI/Amp, were digested with BamHI and XbaI restriction enzyme andligated. The ligation mixture was transformed into E. coli strain SURE(available from Stratagene Cloning Systems, 11099 North Torrey PinesRoad, La Jolla, Calif. 92037) the transformed culture was plated onampicillin media plates and resistant colonies were selected. PlasmidDNA was isolated from transformants and examined by restriction analysisfor the presence of the correct fragment. For expression of therecombinant human TIMP-4, COS cells were transfected with the expressionvector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,(1989)). The expression of the human TIMP-4 HA protein was detected byradiolabelling and immunoprecipitation method (E. Harlow, D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)). Cells were labelled for 8 hours with ³⁵S-cysteine two days posttransfection. Culture media were then collected and cells were lysedwith detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40,0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)).Both cell lysate and culture media were precipitated with a HA specificmonoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGEgels.

EXAMPLE 3

[0388] Cloning and Expression of TIMP-4 Using the Baculovirus ExpressionSystem

[0389] The DNA sequence encoding the full length TIMP-4 protein, ATCC #75946, was amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ sequences of the gene:

[0390] The 5′ primer has the sequence 5′ GCCAGAGGATCCATGCCTGGGAGCCCTCGGCCC 3′ (SEQ ID NO:7) and contains a BamHI restriction enzymesite (in bold) just behind the first 21 nucleotides of the TIMP-4 gene(the initiation codon for translation “ATG” is underlined).

[0391] The 3′ primer has the sequence 5′ CGGCTTCTAGAACTAGGGCTGAACGATGTCAAC 3′ (SEQ ID NO:8) and contains the cleavage site for therestriction endonuclease XbaI and 18 nucleotides complementary to the 3′non-translated sequence of the TIMP-4 gene. The amplified sequences wereisolated from a 1% agarose gel using a commercially available kit(“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment was thendigested with the endonucleases BamHI and XbaI and then purified againon a 1% agarose gel. This fragment is designated F2.

[0392] The vector pA2 (modification of pVL941 vector, discussed below)is used for the expression of the TIMP-4 protein using the baculovirusexpression system (for review see: Summers, M. D. and Smith, G. E. 1987,A manual of methods for baculovirus vectors and insect cell cultureprocedures, Texas Agricultural Experimental Station Bulletin No. 1555).This expression vector contains the strong polyhedrin promoter of theAutographa califomica nuclear polyhidrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BamHI and XbaI.The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusesthe beta-galactosidase gene from E.coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

[0393] The plasmid was digested with the restriction enzymes BamHI andXbaI. The DNA was then isolated from a 1% agarose gel using thecommercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).This vector DNA is designated V2.

[0394] Fragment F2 and the plasmid V2 were ligated with T4 DNA ligase.E.coli HB101 cells were then transformed and bacteria identified thatcontained the plasmid (pBacTIMP-4) with the TIMP-4 gene using theenzymes BamHI and XbaI. The sequence of the cloned fragment wasconfirmed by DNA sequencing.

[0395] 5 micrograms of the plasmid pBacTIMP-4 was co-transfected with1.0 microgram of a commercially available linearized baculovirus(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.) using thelipofection method (Felgner et al. Proc. Natl. Acad. Sci. USA,84:7413-7417 (1987)).

[0396] One microgram of BaculoGold™ virus DNA and 5 micrograsm of theplasmid pBacTIMP-4 were mixed in a sterile well of a microtiter platecontaining 50 microliters of serum free Grace's medium (LifeTechnologies Inc., Gaithersburg, Md.). Afterwards 10 microlitersLipofectin plus 90 microliters Grace's medium were added, mixed andincubated for 15 minutes at room temperature. Then the transfectionmixture was added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace's medium withoutserum. The plate was rocked back and forth to mix the newly addedsolution. The plate was then incubated for 5 hours at 27 degree C. After5 hours the transfection solution was removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum was added.The plate was put back into an incubator and cultivation continued at 27degree C. for four days.

[0397] After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0398] Four days after the serial dilution, the viruses were added tothe cells and blue stained plaques were picked with the tip of anEppendorf pipette. The agar containing the recombinant viruses was thenresuspended in an Eppendorf tube containing 200 microliters of Grace'smedium. The agar was removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus was used to infectSf9 cells seeded in 35 mm dishes. Four days later the supernatants ofthese culture dishes were harvested and then stored at 4 degree C.

[0399] Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-TIMP-4 at a multiplicity of infection (MOI) of 2. Sixhours later the medium was removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 microCi of ³⁵S-methionine and 5 microCi 35S cysteine(Amersham) were added. The cells were further incubated for 16 hoursbefore they were harvested by centrifugation and the labelled proteinsvisualized by SDS-PAGE and autoradiography.

EXAMPLE 4

[0400] Expression Pattern of Human TIMP-4 in Human Tissues

[0401] 20 micrograms of total RNA from each of the above tissues wasdenatured and run on a 1.2% formaldehyde agarose gel and capillaryblotted onto a nylon filter overnight. RNA was immobilized on the filterby UV cross-linking. A random primer probe was prepared from theEcoRI-Xhol insert of the partial TIMP-4 nucleic acid sequence and usedto probe the blot by overnight hybridization in Church buffer with 100μg/ml denatured herring sperm DNA as a blocking agent. Washing wasperformed sequentially with 2× SSC/0.1% SDA and 0.2× SSC/0.1% SDS at 65degrees Celsius.

EXAMPLE 5

[0402] Expression via Gene Therapy

[0403] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37 degree C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0404] pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked bythe long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0405] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer contains an EcoRI site and the 3′primer further includes a HindIII site. Equal quantities of the Moloneymurine sarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

[0406] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0407] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his.

[0408] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

EXAMPLE 6

[0409] Production of an Antibody

[0410] a) Hybridoma Technology

[0411] The antibodies of the present invention can be prepared by avariety of methods. (See, Current Protocols, Chapter 2.) As one exampleof such methods, cells expressing TIMP-4 are administered to an animalto induce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of TIMP-4 protein is prepared andpurified to render it substantially free of natural contaminants. Such apreparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

[0412] Monoclonal antibodies specific for protein TIMP-4 are preparedusing hybridoma technology. (Kohler et al., Nature 256:495 (1975);Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J.Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies andT-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, ananimal (preferably a mouse) is immunized with TIMP-4 polypeptide or,more preferably, with a secreted TIMP-4 polypeptide-expressing cell.Such polypeptide-expressing cells are cultured in any suitable tissueculture medium, preferably in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 g/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.

[0413] The splenocytes of such mice are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP20), available fromthe ATCC. After fusion, the resulting hybridoma cells are selectivelymaintained in HAT medium, and then cloned by limiting dilution asdescribed by Wands et al. (Gastroenterology 80:225-232 (1981). Thehybridoma cells obtained through such a selection are then assayed toidentify clones which secrete antibodies capable of binding the TIMP-4polypeptide.

[0414] Alternatively, additional antibodies capable of binding to TIMP-4polypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the TIMP-4protein-specific antibody can be blocked by TIMP-4. Such antibodiescomprise anti-idiotypic antibodies to the TIMP-4 protein-specificantibody and are used to immunize an animal to induce formation offurther TIMP-4 protein-specific antibodies.

[0415] For in vivo use of antibodies in humans, an antibody is“humanized”. Such antibodies can be produced using genetic constructsderived from hybridoma cells producing the monoclonal antibodiesdescribed above. Methods for producing chimeric and humanized antibodiesare known in the art and are discussed infra. (See, for review,Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533;Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984);Neuberger et al., Nature 314:268 (1985).)

[0416] b) Isolation of Antibody Fragments Directed Against TIMP-4 from aLibrary of scFvs

[0417] Naturally occurring V-genes isolated from human PBLs areconstructed into a library of antibody fragments which containreactivities against TIMP-4 to which the donor may or may not have beenexposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein byreference in its entirety).

[0418] Rescue of the Library.

[0419] A library of scFvs is constructed from the RNA of human PBLs asdescribed in PCT publication WO 92/01047. To rescue phage displayingantibody fragments, approximately 109 E. coli harboring the phagemid areused to inoculate 50 ml of 2xTY containing 1% glucose and 100 μg/ml ofampicillin (2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Fiveml of this culture is used to innoculate 50 ml of 2xTY-AMP-GLU, 2×108 TUof delta gene 3 helper (M13 delta gene III, see PCT publication WO92/01047) are added and the culture incubated at 37° C. for 45 minuteswithout shaking and then at 37° C. for 45 minutes with shaking. Theculture is centrifuged at 4000 r.p.m. for 10 min. and the pelletresuspended in 2 liters of 2xTY containing 100 jig/ml ampicillin and 50ug/ml kanamycin and grown overnight. Phage are prepared as described inPCT publication WO 92/01047.

[0420] M13 delta gene III is prepared as follows: M13 delta gene IIIhelper phage does not encode gene III protein, hence the phage(mid)displaying antibody fragments have a greater avidity of binding toantigen. Infectious M13 delta gene III particles are made by growing thehelper phage in cells harboring a pUC19 derivative supplying the wildtype gene III protein during phage morphogenesis. The culture isincubated for 1 hour at 37° C. without shaking and then for a furtherhour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400r.p.m. for 10 min), resuspended in 300 ml 2xTY broth containing 100 jigampicillin/ml and 25 μg kanamycin/ml (2xTY-AMP-KAN) and grown overnight,shaking at 37° C. Phage particles are purified and concentrated from theculture medium by two PEG-precipitations (Sambrook et al., 1990),resuspended in 2 ml PBS and passed through a 0.45 μm filter (MinisartNML; Sartorius) to give a final concentration of approximately 1013transducing units/ml (ampicillin-resistant clones).

[0421] Panning of the Library.

[0422] Immunotubes (Nunc) are coated overnight in PBS with 4 ml ofeither 100 μg/ml or 10 μg/ml of a polypeptide of the present invention.Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and thenwashed 3 times in PBS. Approximately 1013 TU of phage is applied to thetube and incubated for 30 minutes at room temperature tumbling on anover and under turntable and then left to stand for another 1.5 hours.Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS.Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15minutes on an under and over turntable after which the solution isimmediately neutralized with 0.5 ml of 1.0 M Tris-HCl, pH 7.4. Phage arethen used to infect 10 ml of mid-log E. coli TG1 by incubating elutedphage with bacteria for 30 minutes at 37° C. The E. coli are then platedon TYE plates containing 1% glucose and 100 μg/ml ampicillin. Theresulting bacterial library is then rescued with delta gene 3 helperphage as described above to prepare phage for a subsequent round ofselection. This process is then repeated for a total of 4 rounds ofaffinity purification with tube-washing increased to 20 times with PBS,0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

[0423] Characterization of Binders. Eluted phage from the 3rd and 4throunds of selection are used to infect E. coli HB 2151 and soluble scFvis produced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing.

EXAMPLE 7

[0424] Adenoviral Mediated Gene Therapy

[0425] Adenoviral expression constructs were used to express human andrat TIMP-4 polypeptides in rat thoracic aorta smooth muscle cells andhuman and porcine coronary artery smooth muscle cells in vitro, as wellas in an in vivo rat model of carotid artery balloon injury.

[0426] Adenoviral Constructions:

[0427] The human cDNA sequence encoding the full length TIMP-4 protein,contained in ATCC Deposit No. 75946, was isolated by PCR from cloneHGFAM58 using oligonucleotide primers corresponding to the 5′ and 3′sequence of the gene. The 5′ primer has the sequence 5′CCGGAATTCCACCATGCCTGGGAGCCCTCG 3′ (SEQ ID NO:9) and contains an EcoRIrestriction enzyme site behind the first 17 nucleotides of the TIMP-4gene. The 3′ primer has the sequence 5′ ATCTTTGGTACCTTTCTAGAACTAGGGCTG3′ (SEQ ID NO:10) and contains a XbaI restriction enzyme site and 14nucleotides complementary to the 3′ non-translated sequence of theTIMP-4 gene.

[0428] The rat cDNA sequence encoding the full length TIMP-4 protein wasisolated by RT-PCR using RNA extracted from rat aortic smooth musclecells. Briefly, 2 μg of total RNA was reverse transcribed in 20 μl finalreaction volume in the presence of the four dNTPs, 100 pmol of pdN6(Boehringer Mannheim, Ingelheim, Germany) and 200IU of reversetranscriptase (Superscript, Promega, France). The reaction was performedat 37° C. for 75 min. Then, the enzyme was denatured at 90° C. for 5min. 2 μl of the reaction was used as a template for PCR using specificoligonucleotide promoters for the rat TIMP-4. The sense primer has thesequence 5′ CCGGAATTCCACCATGCCCTGGAGTCCC3′ (SEQ ID NO:11) and containsan EcoRI restriction enzyme site behind the first 17 nucleotides of theTIMP-4 gene. The reverse primer has the sequence 5′CTAGTCTAGACTAGGGCTGGACGATGTCAA 3′ (SEQ ID NO:12) and contains a XbaIrestriction enzyme site and 24 nucleotides complementary to the 3′non-translated sequence of the TIMP-4 gene.

[0429] The amplified sequences were isolated from a 1% agarose gel usinga commercially available kit (“Qiaquick”, Qiagen, Courtaboeuf, France),and then was EcoRI/Xba I subcloned in a transfer vector containing theCMV promoter. This transfer vector contains the Ad5 1-458 regionfollowed by the CMV enhancer/promoter and a chimeric intron generated bycombining the splice donor from the human beta-globin intron 1 and thesplice acceptor from the IgG intervening sequence obtained from pCIplasmid (Promega, Charbonnieres, France). A poly-linker containing,among others, the recognition sites for the restriction endonucleasesXbaI and EcoRI, was inserted upstream to the bovine growth hormonepolyadenylation site followed by the Ad5 3511-5788 region. This vectorcontains the ampicillin resistance gene.

[0430] The E1/E3-deleted adenoviral vector containing the gene encodingTIMP-4 (named AdTG14854) was obtained by homologous recombination inEscherichia coli BJ (Chartier et al., J. Virol. 70(7): 4805-10 (1996)),between the TIMP-4 transfer vector (named pTG14846 for human andpTG14847 for rat; see FIGS. 3A and 3B) and the adenoviral DNA plasmid(named pTG6624; see FIG. 3F) linearized by ClaI. The adenoviral vectorcontaining human TIMP-4 (pTG14854; FIG. 3C) was deposited at theCollection Nationale de Cultures de Microorganismes, Institute Pasteur(25 Rue du Docteur Roux, F-74724 Paris Cedex 15, France), on Jul. 9,2001, and received deposit registration number CNCM 1-2696.

[0431] Virus propagation, purification and titration of infectious units(iu) by indirect immunofluorescence of the viral DNA binding proteinwere carried out as described previously (Lusky et al., J. Virol.72(3):2022-32 (1998)). Purified virus was stored in viral storage buffer(1 M sucrose, 10 mM Tris-HCl [pH=8.5], 1 mM MgCl₂, 150 mM NaCl, 0.005%[vol/vol] Tween 80). Bacteria comprising DNA plasmid containing humanTIMP-4.

[0432] Cells and Culture Conditions:

[0433] Rat thoracic aorta smooth muscle cells were isolated from normalrats (ratAoSMCs) and from injured rats 15 days after balloon catheterdeendothelialization (ratIT5) by enzymatic digestion as previouslydescribed (Orlandi at al., Arterioscler. Thromb. 14(6):982-9 (1994)).Porcine coronary artery SMCs were isolated from normal pigs (pigCoSMCs)and from injured animals 15 days after stent placement (pigIT15) byenzymatic digestion as previously described (Christen et al., Circ. Res.85(1):99-107 (1999)). The human coronary artery SMCs were purchased fromClonetics (Walkersville, Md., USA). Rat and pig cells were cultured inDMEM containing 10% FCS (Life Technologies, Cergy-Pontoise, France).Human cells were cultured in SMGM2 medium containing 5% FCS(Bioproducts, Gagny, France).

[0434] Adenoviral Cell Infection:

[0435] SMCs were infected in suspension at an MOI corresponding to 80%of infected cells. Briefly, cells were trypsinized, centrifuged and thenresuspended in 2% FCS cell culture medium (5×10⁶ cells in 500 μl). Thevirus was added for a 30 min. incubation time at 37° C., 5% CO2. Cellswere rinsed in fresh medium and finally resuspended and plated in 10% or5% FCS corresponding medium.

[0436] Gelatin Zymography:

[0437] Recombinant human MMP2 was purchased from R&D System (Oxon, UK)and used at 2.5 ng in the gel. Briefly, cell lysates (from 2×10⁵ cells)were mixed with Novex tris-Glycine Sample Buffer and let stand 10minutes at room temperature. Samples (20 μl) were then subjected toelectrophoresis on 10% Tris-Glycine gel with 0.1% gelatine incorporatedas a substrate. Gels were washed in Novex Renaturing Buffer with gentleagitation for 30 min. at room temperature. Renaturing Buffer is thendecanted and replaced with Developing Buffer for a 4 hour incubation at37° C. Gels were stained with Coomassie Blue R-250 for 30 min.Metalloproteinase produced clear areas of lysis in the gel.

[0438] Rat Carotid Artery Balloon Injury Model:

[0439] Adult male Wistar rats (body weight >400g) were used forexperiments (Iffa-Credo). Anesthesia was induced with intraperitonealinjection of Ketamine (Imalgene, Rhône-Mérieux, Lyon, France) andAcepromazin (Vetranquil 0.5%, Sanofi, Libourne, France) in doses of 23.1and 3.84 mg/kg respectively. Animals were anticoagulated withintravenous injection of 200 U/kg of human heparin (Choay, SanofiWinthrop, Gentilly, France). The left common carotid artery wassurgically exposed and an arteriotomy was made on the left externalcarotid artery. Deendothelialization was achieved by three passages of a2F Fogarty balloon catheter (Baxter, Maurepas, France) filled with 0.2ml air. A 1 cm length segment of the carotid was isolated withmicrosurgical clamps and a 24-gauge catheter was introduced through thearteriotomy. The segment was flushed with 0.2 ml NaCl 0.9% and 50 μl ofadenoviral solution (2×10⁹ iu) was infused. The solution was allowed todwell in the carotid for 5 minutes during which the carotid segmentremained distended. The solution was withdrawn, the external carotidartery was ligated and blood flow was reestablished through the commonand the internal carotid arteries. Rats were sacrificed at D14 postinjury. After lethal pentobarbital injection and cannulation of theheart, vessels were perfused with lx PBS solution and perfusion-fixedeither with 2% or 4% formaldehyde in PBS at normal blood pressure. Then,carotids were excised and treated for histological analyses.

[0440] For morphometric analysis, carotids were fixed in 4% formaldehydeand embedded in paraffin. Five μm sections were stained either withhematoxylin or with hematoxylin and eosin and the media and intimalarea, as well as medial and neointimal cell number were evaluated byimage analysis on 3 cross sections for each vessel (NIH Image software).

[0441] TIMP-4 mRNA Expression

[0442] Total RNA was extracted (RNA Now reagent, Ozyme, Montigny,France) at 24 and 48 hours after AdTG14854 (CMV-human TIMP-4; see FIG.3C) infection of human and porcine CaSMCs, and AdTG14855 (CMV-ratTIMP-4; see FIG. 3D) infection of rat AoSMCs. The presence of the mRNAwas detected by Northern blot. Briefly, 10 μg of total RNA extractedfrom each of the above cell populations were denatured and run on a 1%formaldehyde agarose gel and capillary blotted onto a Hybond nylonmembrane overnight. RNA was fixed on the membrane by heating at 80° C.for 2 hours. A random primer probe (Amersham Multi Prime Kit) wasprepared from the SmaI-KpnI insert of the partial TIMP-4 nucleic acidsequence and used to probe the blot by 3 hour hybridization in Amershamhybridization buffer containing 200 μg/ml denatured herring sperm DNA asa blocking agent. Washing was performed sequentially with 1× SSC-0.1%SDS (2×15 min.) and 0.1× SSC-0.1% SDS (1×10 min.). Results indicate thatthe exogeneous TIMP-4 mRNA is present in infected SMC of human andporcine coronary arteries and rat aorta. In all cell types, two majorbands were observed in agreement with published data (Gomez, EuropeanJournal of Cell Biology, 74:111 (1997)).

[0443] TIMP-4 Protein Expression

[0444] Total proteins were extracted from cells and culture supernatants2 days, 3 days, 5 days and 7 days after AdTG14854 infection of human andporcine CaSMCs, and AdTG14855 infection of rat AoSMCs. The presence ofTIMP-4 protein was detected by Western blot. Briefly, 150 μg ofextracted proteins were run on a 10% Nupage gel (Novex, Invitrogen,Groningen, the Netherlands), and then blotted on a nitrocellulosemembrane (Novex). The membrane was incubated in a blocking solution(PBS-2% milk) overnight before detection of the blotted antigen, usingan anti-TIMP-4 antibody (clone S720, Abcam, Cambridge, UK). Western blotdetection of the TIMP-4 protein indicates that TIMP-4 is present inlarge amounts in human coronary cell supernatants and in lower amountsin pig cell supernatants. In rat SMC supernatants, no protein wasdetected in IT15 and only a faint signal in ratAo. These resultsindicate either that the protein is not equally expressed by all celltypes or that the antibody does not recognize the rat TIMP-4. Byinfecting human cells with the rat TIMP-4 adenovirus and the rat cellswith the human TIMP-4 adenovirus, it was observed by western blot thatthe production was equivalent in the different cell types for a definedvector suggesting that the antibody is specific for the human protein.TIMP-4 activity

[0445] Staining of gelatin zymogram gels revealed a gelatin lysisactivity (MMP activity) with cell lysates corresponding to the Ad-nullinfection whereas, no areas of gel lysis were observed with cell lysatefrom Ad-TIMP-4 infected cells. Taken together with Western blot resultsshowing an equal amount of MMP2 in cells infected either with Ad-null orAd-TIMP-4 (data not shown), the zymogram data indicated that thedecrease of MMP activity was due to the TIMP-4 inhibitory effect.

[0446] Effects on Cell Proliferation

[0447] SMC proliferation was studied in SMCs infected in suspension atan MOI corresponding to 80% of infected cells. The results indicate thaton rat and pig cells, TIMP-4 is not able to significantly inhibit cellgrowth. On human cells, an inhibitory effect was observed which could bedue to adenovirus toxicity. To confirm this hypothesis, lower MOIs ofAdTIMP-4 were used to infect human cells. The results are summarized inTable Table 1: TABLE 1 MOI adenovirus MOI 0 MOI 1 MOI 10 MOI 50 MOI 100300 AdTG6401 100%  75% 75% 67% 44% 32.7% AdTG14854 100% 123% 84% 49% 30%21.4%

[0448] An antiproliferative effect was observed at MOIs starting fromMOI 50. At this and higher MOIs a toxic effect of the Adnull itself anda slight additional toxicity of the TIMP-4 was observed. In conclusion,these data suggest that TIMP-4 has no growth inhibitory effect on rat,pig and human SMCs except at very high adenoviral load. These resultsare in agreement with published reports on the effects of other membersof the TIMP family.

[0449] Effects on Cell Migration

[0450] The assay of migration in matrigel drops was used. TIMP-4infected Human CaSMCs were incorporated into 50 μl of matrigel. Anon-selective MMP inhibitor (doxycycline) was added at differentconcentrations in the aim to inhibit most of the non-gelatinase(collagenase) activity. In addition, the medium was changed after 24hours to remove soluble MMPs. Four days after seeding, an anti-migratoryactivity of TIMP-4 was observed. This effect is gelatinase-dependentsince TIMP-4 and doxycycline have cummulative effects. This experimentwas repeated twice with the same result. TIMP-4 inhibition of neointimalthickening in the rat injured carotid model

[0451] To examine the effect of Ad-TIMP-4 infection on neointimalformation, 12 rats were infected after injury with 2×10⁹ IU of AdTG14855or AdTG6401. The carotids of these animals were collected at 14 daysafter injury and both neointimal and medial areas were measured (seeTable 2). There was a significant 74% reduction in neointimal area inAd-TIMP-4 infected vessels (1.04+/−0.32 mm2; p=0.00018; n=6) comparedwith Ad-null infected vessels (5.03+/−1.66 mm²; n=6). No significantdifference was seen in medial area (5.86+/−0.32 versus 6.14+/−0.65 forAdTIMP-4 and Ad-Null infected vessels, respectively). As expected, theratio of neointima to media showed a significant difference betweenAd-TIMP-4 and Ad-Null infected vessels (0.18+/−0.05 versus 0.80+/−0.16;p=0.01). These results show that adenovirus-mediated gene transfer ofrat TIMP-4 to the rat carotid artery immediately after injury, causes asignificant decrease in neointima development. TABLE 2 AdTG6401 (2 × 10⁹iu) AdTG14855 (2 × 10⁹ iu) Lumen area 13.81 ± 2.10  15.77 ± 1.84 (p =0.11) Media area 6.14 ± 0.65  5.86 ± 0.32 (p = 0.37) Intima area 5.03 ±1.66  1.04 ± 0.32 (p = 0.00018) Neointima/media 0.80 ± 0.16  0.18 ± 0.05(p = 0.000012) Lumen perimeter 13.88 ± 0.88  15.25 ± 0.59 (p = 0.01)

[0452]

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 8 <210> SEQ ID NO 1 <211>LENGTH: 675 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(672) <221> NAME/KEY:sig_peptide <222> LOCATION: (1)..(87) <221> NAME/KEY: mat_peptide <222>LOCATION: (88)..() <400> SEQUENCE: 1 atg cct ggg agc cct cgg ccc gcg ccaagc tgg gtg ctg ttg ctg cgg 48 Met Pro Gly Ser Pro Arg Pro Ala Pro SerTrp Val Leu Leu Leu Arg -25 -20 -15 ctg ctg gcg ttg ctg cgg ccc ccg gggctg ggt gag gca tgc agc tgc 96 Leu Leu Ala Leu Leu Arg Pro Pro Gly LeuGly Glu Ala Cys Ser Cys -10 -5 -1 1 gcc ccg gcg cac cct cag cag cac atctgc cac tcg gca ctt gtg att 144 Ala Pro Ala His Pro Gln Gln His Ile CysHis Ser Ala Leu Val Ile 5 10 15 cgg gcc aaa atc tcc agt gag aag gta gttccg gcc agt gca gac cct 192 Arg Ala Lys Ile Ser Ser Glu Lys Val Val ProAla Ser Ala Asp Pro 20 25 30 35 gct gac act gaa aaa atg ctc cgg tat gaaatc aaa cag ata aag atg 240 Ala Asp Thr Glu Lys Met Leu Arg Tyr Glu IleLys Gln Ile Lys Met 40 45 50 ttc aaa ggg ttt gag aaa gtc aag gat gtt cagtat atc tat acg cct 288 Phe Lys Gly Phe Glu Lys Val Lys Asp Val Gln TyrIle Tyr Thr Pro 55 60 65 ttt gac tct tcc ctc tgt ggt gtg aaa cta gaa gccaac agc cag aag 336 Phe Asp Ser Ser Leu Cys Gly Val Lys Leu Glu Ala AsnSer Gln Lys 70 75 80 cag tat ctc ttg act ggt cag gtc ctc agt gat gga aaagtc ttc atc 384 Gln Tyr Leu Leu Thr Gly Gln Val Leu Ser Asp Gly Lys ValPhe Ile 85 90 95 cat ctg tgc aac tac atc gag ccc tgg gag gac ctg tcc ttggtg cag 432 His Leu Cys Asn Tyr Ile Glu Pro Trp Glu Asp Leu Ser Leu ValGln 100 105 110 115 agg gaa agt ctg aat cat cac tac cat ctg aac tgt ggctgc caa atc 480 Arg Glu Ser Leu Asn His His Tyr His Leu Asn Cys Gly CysGln Ile 120 125 130 acc acc tgc tac aca gta ccc tgt acc atc tcg gcc cctaac gag tgc 528 Thr Thr Cys Tyr Thr Val Pro Cys Thr Ile Ser Ala Pro AsnGlu Cys 135 140 145 ctc tgg aca gac tgg ctg ttg gaa cga aag ctc tat ggttac cag gct 576 Leu Trp Thr Asp Trp Leu Leu Glu Arg Lys Leu Tyr Gly TyrGln Ala 150 155 160 cag cat tat gtc tgt atg aag cat gtt gac ggc acc tgcagc tgg tac 624 Gln His Tyr Val Cys Met Lys His Val Asp Gly Thr Cys SerTrp Tyr 165 170 175 cgg ggc cac ctg cct ctc agg aag gag ttt gtt gac atcgtt cag ccc 672 Arg Gly His Leu Pro Leu Arg Lys Glu Phe Val Asp Ile ValGln Pro 180 185 190 195 tag 675 <210> SEQ ID NO 2 <211> LENGTH: 224<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 Met ProGly Ser Pro Arg Pro Ala Pro Ser Trp Val Leu Leu Leu Arg -25 -20 -15 LeuLeu Ala Leu Leu Arg Pro Pro Gly Leu Gly Glu Ala Cys Ser Cys -10 -5 -1 1Ala Pro Ala His Pro Gln Gln His Ile Cys His Ser Ala Leu Val Ile 5 10 15Arg Ala Lys Ile Ser Ser Glu Lys Val Val Pro Ala Ser Ala Asp Pro 20 25 3035 Ala Asp Thr Glu Lys Met Leu Arg Tyr Glu Ile Lys Gln Ile Lys Met 40 4550 Phe Lys Gly Phe Glu Lys Val Lys Asp Val Gln Tyr Ile Tyr Thr Pro 55 6065 Phe Asp Ser Ser Leu Cys Gly Val Lys Leu Glu Ala Asn Ser Gln Lys 70 7580 Gln Tyr Leu Leu Thr Gly Gln Val Leu Ser Asp Gly Lys Val Phe Ile 85 9095 His Leu Cys Asn Tyr Ile Glu Pro Trp Glu Asp Leu Ser Leu Val Gln 100105 110 115 Arg Glu Ser Leu Asn His His Tyr His Leu Asn Cys Gly Cys GlnIle 120 125 130 Thr Thr Cys Tyr Thr Val Pro Cys Thr Ile Ser Ala Pro AsnGlu Cys 135 140 145 Leu Trp Thr Asp Trp Leu Leu Glu Arg Lys Leu Tyr GlyTyr Gln Ala 150 155 160 Gln His Tyr Val Cys Met Lys His Val Asp Gly ThrCys Ser Trp Tyr 165 170 175 Arg Gly His Leu Pro Leu Arg Lys Glu Phe ValAsp Ile Val Gln Pro 180 185 190 195 <210> SEQ ID NO 3 <211> LENGTH: 33<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: 5′ TIMP-4 primer with BamH1site <400> SEQUENCE: 3gccagaggat cctgcagctg cgccccggcg cac 33 <210> SEQ ID NO 4 <211> LENGTH:33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: 3′ TIMP-4 primer with XbaI site <400> SEQUENCE:4 cggcttctag aactagggct gaacgatgtc aac 33 <210> SEQ ID NO 5 <211>LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: 5′ TIMP-4 primer with BamHI site <400>SEQUENCE: 5 gccagaggat ccgccaccat gcctgggagc cctcggccc 39 <210> SEQ IDNO 6 <211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: 3′ TIMP-4 primer withXbaI site <400> SEQUENCE: 6 cggcttctag aatcaagcgt agtctgggac gtcgtatgggtagggctgaa cgatgtcaac 60 <210> SEQ ID NO 7 <211> LENGTH: 33 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: 5′ TIMP-4 primer with BamHI site <400> SEQUENCE: 7gccagaggat ccatgcctgg gagccctcgg ccc 33 <210> SEQ ID NO 8 <211> LENGTH:33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: 3′ TIMP-4 primer with XbaI site <400> SEQUENCE:8 cggcttctag aactagggct gaacgatgtc aac 33

What is claimed is:
 1. An isolated polynucleotide comprising a memberselected from the group consisting of: (a) a polynucleotide encoding thepolypeptide as set forth in SEQ ID NO:2; (b) a polynucleotide capable ofhybridizing to and which is at least 70% identical to the polynucleotideof (a); and (c) a polynucleotide fragment of the polynucleotide of (a)or (b).
 2. The polynucleotide of claim 1 wherein the polynucleotide isDNA.
 3. The polynucleotide of claim 2 which encodes the polypeptide asset forth in SEQ ID NO:2.
 4. The polynucleotide of claim 2 which encodesthe polypeptide comprising amino acid 1 to 195 of of SEQ ID NO:2.
 5. Thepolynucleotide of claim 2 comprising the nucleotide sequence of SEQ IDNO:1 from nucleotide 1 to
 675. 6. An isolated polynucleotide comprisinga member selected from the group consisting of: (a) a polynucleotidewhich encodes a mature polypeptide encoded by the DNA contained in ATCCDeposit No. 75946; (b) a polynucleotide which encodes a polypeptideexpressed by the DNA contained in ATCC Deposit No. 75946; (c) apolynucleotide capable of hybridizing to and which is at least 70%identical to the polynucleotide of (a) or (b); and (d) a polynucleotidefragment of the polynucleotide of (a), (b) or (c).
 7. A vectorcontaining the DNA of claim
 2. 8. A host cell genetically engineeredwith the vector of claim
 7. 9. A process for producing a polypeptidecomprising: expressing from the host cell of claim 8 the polypeptideencoded by said DNA.
 10. A process for producing cells capable ofexpressing a polypeptide comprising transforming or transfecting thecells with the vector of claim
 7. 11. A polypeptide comprising a memberselected from the group consisting of (i) a polypeptide having thededuced amino acid sequence of SEQ ID NO:2 and fragments, analogs andderivatives thereof; (ii) a polypeptide comprising amino acid 1 to aminoacid 195 of SEQ ID NO:2; and (iii) a polypeptide encoded by the cDNA ofATCC Deposit No. 75946 and fragments, analogs and derivatives of saidpolypeptide.
 12. A compound effective as an agonist for the polypeptideof claim
 11. 13. A compound effective as an antagonist against thepolypeptide of claim
 11. 14. A method for the treatment of a patienthaving need of TIP-4 comprising: administering to the patient atherapeutically effective amount of the polypeptide of claim
 11. 15. Themethod of claim 14 wherein said therapeutically effective amount of thepolypeptide is administered by providing to the patient DNA encodingsaid polypeptide and expressing said polypeptide in vivo.
 16. A methodfor the treatment of a patient having need of TIMP-4 comprising:administering to the patient a therapeutically effective amount of thecompound of claim
 12. 17. A method for the treatment of a patient havingneed to inhibit TIMP-4 comprising: administering to the patient atherapeutically effective amount of the antagonist of claim
 13. 18. Aprocess for identifying compounds active as agonists or antagonists tothe polypeptide of claim 11 comprising: combining an MMP, human TIMP-4,a compound to be screened and a reaction mixture containing substratecapable of degradation by the MMP, wherein said substrate is labeled;and determining the ability of the compound to enhance or block thedegradation of the substrate by said MMP by measuring the label releasedfrom the substrate.
 19. A process for diagnosing a disease or asusceptibility to a disease related to a mutation in the polypeptide ofclaim 11 comprising: determining a mutation in the human TIMP-4 nucleicacid sequence.
 20. A diagnostic process comprising: analyzing for thepresence of the polypeptide of claim 11 in a sample derived from a host.21. A method of treating restenosis in a patient, comprisingadministering to the patient an isolated nucleic acid molecule encodinga TIMP-4 polypeptide selected from the group consisting of: (a) theamino acid sequence shown as residues—29 to 195 in SEQ ID NO:2; (b) theamino acid sequence shown as residues—28 to 195 in SEQ ID NO:2; (c) theamino acid sequence shown as residues 1 to 195 in SEQ ID NO:2; and (d) afragment of the sequence described in (a) whereing a polypeptideconsisting of the fragment retains protease inhibiting activity; whereinsaid nucleic acid molecule is operatively linked to a transcriptioncontrol sequence; and wherein the expression of said nucleic acidmolecule results in an increased amount of the TIMP-4 polypeptide in anamount effective to inhibit metalloproteinase activity.
 22. The methodof claim 21, wherein said isolated nucleic acidmolecule is administeredto said patient in a viral vector delivery vehicle.
 23. The method ofclaim 22, wherein said viral vector delivery vehicle is from adenovirus.